Methods of use of inhibitors of phosphodiesterases and modulators of nitric oxide, reactive oxygen species, and metalloproteinases in the treatment of peyronie&#39;s disease, arteriosclerosis and other fibrotic diseases

ABSTRACT

The present methods and compositions are of use for treatment of conditions involving fibrosis, such as Peyronie&#39;s disease plaque, penile corporal fibrosis, penile veno-occlusive dysfunction, Dupuytren&#39;s disease nodules, vaginal fibrosis, clitoral fibrosis, female sexual arousal disorder, abnormal wound healing, keloid formation, general fibrosis of the kidney, bladder, prostate, skin, liver, lung, heart, intestines or any other localized or generalized fibrotic condition, vascular fibrosis, arterial intima hyperplasia, atherosclerosis, arteriosclerosis, restenosis, cardiac hypertrophy, hypertension or any condition characterized by excessive fibroblast or smooth muscle cell proliferation or deposition of collagen and extracellular matrix in the blood vessels and/or heart. In certain embodiments, the compositions may comprise a PDE-4 inhibitor, a PDE-5 inhibitor, a compound that elevates cGMP and/or PKG, a stimulator of guanylyl cyclase and/or PKG, a combination of a compound that elevates cGMP, PKG or NO with an antioxidant that decreases ROS, or a compound that increases MMP activity.

CROSS-REFERENCE TO RELATED APPLICATION

The application is a continuation of co-pending U.S. patent applicationSer. No. 15/832,918, filed Dec. 6, 2017, which is a continuation of U.S.patent application Ser. No. 15/277,550, filed Sep. 27, 2016, which is acontinuation of U.S. patent application Ser. No. 14/453,543, filed Aug.6, 2014, which is a continuation of U.S. patent application Ser. No.13/372,094, filed Feb. 13, 2012, which is a continuation of U.S. patentapplication Ser. No. 10/779,069, filed Feb. 13, 2004 (issued as U.S.Pat. No. 8,133,903), which claims the benefit under 35 U.S.C. § 119(e)of U.S. Provisional Application No. 60/420,281, filed on Oct. 22, 2002,and claims the benefit of International Application No. PCT/US03/33400,filed on Oct. 21, 2003.

BACKGROUND OF THE INVENTION Field

The present methods and compositions relate to the field of Peyronie'sdisease, arteriosclerosis and other fibrotic conditions. Moreparticularly, the method and compositions concern use ofphosphodiesterase (PDE) inhibitors and modulators of nitric oxide,reactive oxygen species and metalloproteinases in the treatment of suchconditions. In particular embodiments, the inhibitors inhibit type 4and/or type 5 PDEs.

Description of Related Art

Peyronie's disease (PD) is a fibromatosis (Hellstrom and Bivalacqua,2000; Schwarzer et al., 2001; Jarow et al., 1997; Devine et al., 1997)of the tunica albuginea (TA), the specialized lining of the corporacavernosa of the penis. Clinically, this usually leads to peniledeformation (curved penis during erection), pain, and quite frequentlyerectile dysfunction. The initiating event is believed to be an externalforce to the erect penis that results in an injury to the TA of thecorpora and the TA fails to heal normally (Jarow et al., 1997; Devine etal., 1997; Diegelmann, 1997; Sherratt and Dallon, 2002). In thedetumesced state, the only indication of the disease is the palpation ofa knot or scar within the TA, which in its most severe state presents asa calcified plaque.

PD affects about 5% of men in the USA, and translating into about 3-4million affected American males. Although the condition is not alwaysassociated with erectile dysfunction, patients usually present to thephysician with either recognition of a palpable plaque on the penileshaft, pain with tumescence, impotence and/or difficulty withintromission that is due to curvature of the erect penis. Since thedisorder was first described in 1743, no medical treatment has everproven to be beneficial in combating the condition, thereby highlightingthe need to develop novel approaches to combat this disorder.

There may also be a genetic predisposition to developing PD, since it isassociated with other contractures such as Dupuytren's disease (palmarfascia; 10-20% incidence or more in PD) (Connelly, 1999) and Ledeshore'sdisease (plantar fascia). The pathophysiology is characterized bylocalized disruption of the TA, increased microvascular permeability,persistent fibrin (deficient fibrinolysis) and collagen deposition,perivascular inflammation, disorganization and loss of elastic fibers(release of elastase by macrophages), disorganized collagen bundles, andan increase in TGF-β1 synthesis. This represents impairment in therepair process that leads to persistent fibrosis and a loss ofelasticity of the TA.

PD can rarely be alleviated by medical treatment with anti-inflammatoryagents (corticosteroids, antihistamine), antioxidants (vitamin E,superoxide dismutase), collagen breakdown (collagenase), Ca channelblockers (verapamil), and other antifibrotic compounds (colchicine,Potaba: K aminobenzoate) (Hellstrom and Bivalacqua, 2000). In mostcases, surgery is the only available option to correct the deformity andalleviate the pain so that normal sexual activity can be resumed. A needexists for non-surgical methods of treatment of Peyronie's disease andother medical conditions in which fibrosis is important.

Fibrotic disease is not limited to the reproductive organs, but can befound in other tissues, such as cardiovascular tissues. Both erectiledysfunction (ED) and cardiovascular disease, particularly hypertension,are prevalent in the aging male (Kloner et al., 2002; Sullivan et al.,2001; Melman et al., 1999). One of the underlying causes of hypertensionis arteriosclerosis, or arterial stiffness, due to an acquired fibrosisof the media of the arterial wall (Breithaupt-Grogler and Belz, 1999;Robert, 1999; Intengan and Schiffrin, 2000, 2001; Fornieri et al.,1992). Arteriosclerosis is significantly associated with aging, and isrecognized by an increase in collagen, and in some cases by a loss ofsmooth muscle cells (SMC) within the arterial media, which results in adecrease in the SMC/collagen ratio, often accompanied by endothelialdysfunction (Cai and Harrison, 2000).

The pathogenesis of aging associated ED, both in the human and rat, ismostly related to the loss of SMC in the penile corpora cavernosa byapoptosis, with a corresponding increase in collagen fibers (Melman andGingell, 1999; Cai and Harrison, 2000; Melman, 2001; Garban et al.,1995; Ferrini et al., 2001a). The clinical result of this aging processin the penis is defective cavernosal SMC relaxation leading toveno-occlusive dysfunction (Breithaupt-Grogler and Belz, 1999; Rogers etal., 2003), the most common cause of ED.

In the arterial tree, excessive collagen deposition in the media, withor without loss of SMC, leads to defective vaso-relaxation andclinically may present as hypertension (Breithaupt-Grogler and Belz,1999; Robert, 1999; Intengan and Schiffrin, 2000, 2001). Because thepenis may be considered a specialized extension of the vascular tree,the common alterations observed in the SMC of both the penis andperipheral vascular system in the aging male, leading to ED andhypertension, respectively, suggest that both conditions may share acommon etiology.

A need exists for effective methods to treat and/or ameliorate thesymptoms of a variety of fibrotic disease, such as PD, ED andarteriosclerosis. No effective method of treatment currently exists thatis directed towards the molecular pathways underlying excessive collagendeposition.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention fulfill an unresolved needin the art, by providing novel methods for therapeutic treatment ofPeyronie's disease, erectile dysfunction, arteriosclerosis and otherfibroses. In some embodiments, PD plaques and/or other fibroticconditions can be pharmacologically arrested or reduced in size, bydecreasing collagen synthesis and inducing myofibroblast apoptosis byincreasing the NO/ROS ratio, the levels of cGMP, or the activation ofits effector, PKG in the TA and/or stimulating collagen degradation byactivating the MMPs and/or down-regulating the expression of the MMPinhibitors (TIMP), by increasing NO/cGMP levels and/or the thymosins inthe TA.

Particular embodiments of the invention may be directed towardsincreasing levels of cGMP and/or cAMP by selective inhibition ofphosphodiesterase (PDE) isoforms. PDE isoforms of interest in the TA andin PD plaque tissues include PDE5A-3, PDE4A, PDE4B and PDE4D. Asnon-limiting examples, pentoxifylline and similar compounds act as anon-specific cAMP-PDE inhibitor and increase cAMP levels, whilesildenafil and similar compounds selectively inhibit PDE5A and increasecGMP levels.

Other embodiments may involve increasing NO levels, for example byadministering L-arginine, a stimulator of NOS activity. As shown in thefollowing examples, pentoxifylline, sildenafil and L-arginine all act toreduce the expression of collagen I and α-smooth muscle actin. Long-termadministration of nitrergic agents, such as pentoxifylline, sildenafiland L-arginine may be of use to reduce PD plaque size andcollagen/fibroblast ratio and may reverse or prevent the furtherdevelopment of the fibrosis observed in PD. ED, arteriosclerosis andother fibrotic conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the claimedsubject matter. The embodiments may be better understood by reference toone or more of these drawings in combination with the detaileddescription presented herein.

FIG. 1. The effect of NO, TGF-β1, ROS and cGMP on fibroblast/myoblastdifferentiation and collagen deposition.

FIG. 2. Inhibition of collagen deposition in the fibrotic plaque inducedby TGF-β1 in the rat TA, by long-term oral treatment with L-arginine andPDE inhibitors, estimated by Masson staining. (A) Microphotographs (40×)of cross sections of half of the rat penis. Dark arrows indicate theouter extent of plaque development and of tunical thickening. Lightarrowheads (lower right-hand corner of each panel) indicate the site ofTGF-β1 injection. The control (−) was injected with TGF-β1 injection, notreatment was given. L-ARG received a TGF-β1 injection and L-arginine inwater. SIL received a TGF-β1 injection and sildenafil in water. PXFreceived a TGF-β1 injection and pentoxifylline in water. (B) QIAevaluation (n=5 per group). Five sections/animal, three fields/section.

FIG. 3. Stimulation of apoptosis, as estimated by TUNEL, in the fibroticplaque induced by TGF-β1 injection into the rat TA, following oraltreatment with L-arginine, sildenafil or pentoxiphylline. (A)Microphotographs (400×) of tissue sections. Arrows indicate apoptoticcells in the site of the plaque. The control (−) was injected withTGF-β1 injection, no treatment was given. L-ARG received a TGF-β1injection and L-arginine in water. SIL received a TGF-β1 injection andsildenafil in water. PXF received a TGF-β1 injection and pentoxifyllinein water. (B) QIA (n=5 per group) as in FIG. 2.

FIG. 4. Expression of PDE-5 mRNA and protein in the human PD plaque andnormal tunica albuginea, and their homologous tissues in the TGF-β1 ratmodel of PD. (A) Ethidium bromide-stained DNA bands obtained by RT/PCRfrom RNAs isolated from the respective tissues, and fractionated onagarose gels. (B) Luminol-stained protein bands obtained by western bloton polyacrylamide gels. PS: penile shaft; TA: tunica albuginea; PD:Peyronie's disease; CC: corpora cavernosa; CER: cerebellum; CRU: penilecrura. (C) Microphotgraphs (200×) of sections from human and untreatedrat tissues. D.ART: dorsal artery; ART: artery. Arrows show positivecells for PDE-5.

FIG. 5. Inhibition of collagen I synthesis and myofibroblastdifferentiation by PDE inhibitors in fibroblast cultures from a human PDplaque. (A) QIA evaluation of collagen I and ASMA expression. Control(C) no addition; 50SIL: sildenafil (50 nM); 200SIL: sildenafil (200 nM);200PXF:pentoxifylline (200 nM). (B) DAB-stained immunocytochemicaldetection of collagen III in PD cells incubated for 3 days in DME-serumfree medium in the presence or absence of 5 ng/ml of TGF-β1 (200×).

FIG. 6. Effects on collagen I synthesis and myofibroblastdifferentiation in fibroblast cultures from the human PD plaque by acGMP analog (8-Br cGMP), estimated by immunocytochemistry. Cells wereincubated for 3 days with the indicated concentration and collagen I andASMA were immuno-cytochenmically detected. Values are means+/−SEM forthree separate incubations. p<0.05 were as follows: panel A: a vs b,c;panel D: a vs c; all others were non-significant.

FIG. 7. Expression of PDE-5 mRNA and protein in fibroblast cultures fromhuman PD plaque and human and rat normal tunica albuginea. (A) Ethidiumbromide staining of PDE-5A cDNA bands generated from cell RNA by RT-PCRand fractionated on agarose gels. (B) Luminol detection of PDE-5 proteinbands obtained by western blot of cell extracts on PAGE. Arrows indicatePDE-5A variants. DUP: cells from Dupuytren's nodules. (C)Microphotographs (200×) of cell cultures stained with the indicatedantibodies and counter-stained with Meyer haematoxylin

FIG. 8. Gene transfer to the tunica albuginea of plasmid and adenoviralcDNA constructs facilitated by electroporation. Both the pCMV-βgal andthe AdV-CMV-βgal constructs were injected into the tunica albuginea ofthe rat, followed by electroporation, and 10 days later rats weresacrificed and frozen fixed tissue sections were stained with X-gal, andcounterstained with neutral red. TA: tunica albuginea. (200×magnification).

FIG. 9. Induction of TGF-β1 expression in the Peyronie-like plaqueinduced by fibrin in the tunica albuginea of the rat. (A) Sectionsadjacent to the ones for plaques shown on FIG. 11 were immunostainedwith an antibody for TGF-β1. Arrows point to cells with intensestaining. (220× magnification) (B) Image quantitation of positive cellsin the average field of the fibrotic plaque (n=5). Values aremeans+/−SEM, and p values are as indicated. (200× magnification)

FIG. 10. Confirmation by RT/PCR of alterations in the expression ofcertain genes in the human Peyronie's plaque as compared to the normaltunica albuginea.

FIG. 11. PD-like plaque similar to the one induced in the rat can beelicited in the tunica albuginea of the mouse by TGF-β1 injection,detected by Masson staining. Low (4×, top); and high (100×, bottom)magnifications of mouse penis injected with saline and TGF-β1. Lightarrows show the site of TGF-β1 injection. Boxes represent the area ofhigh magnification. CC corpora cavernosa; UR: urethra; TA: tunicaalbuginea; Pl: plaque; NB: nerve bundle.

FIG. 12. Expression of PDE-4 mRNA and protein in the human PD plaque andnormal tunica albuginea, and their homologous tissues in the TGF-β1 ratmodel of PD, and in fibroblasts cultured from these tissues. (A)Ethidium bromide staining of DNA generated from PD and normal human TAtissue by RT/PCR with primers for PDE4A, PDE4B, and GAPDH (referencegene), separated by agarose gel electrophoresis. (B) PDE4 mRNA in ratpenile shaft (PS), rat TA cells, or human TA or PD cells. TA: tunicaalbuginea; PD: Peyronie's disease; CC: corpora cavernosa smooth muscle;PS: penile shaft. (C) Luminol-stained protein bands on western blots ofhuman tissue and cell extracts with the antibody against PDE4A.

FIG. 13. Immunodetection of PDE-4 protein in the rat penis and culturesof rat and human tunica albuginea fibroblasts. Microphotographies oftissue sections (top panels) or cell cultures (middle and bottompanels), as indicated, submitted to immunodetection with antibodiesagainst PDE4A or PDE4D, and counterstained with Meyer haematoxylin.

FIG. 14. Effect of pentoxifylline and sildenafil on cAMP and cGMP levelsin fibroblast cultures from human PD plaque, estimated by enzymeimmunoassays. Cells were incubated for 3 days in fibroblast growthmedium (FGM)/10% fetal bovine serum, in the presence of SNAP (100 uM;medium changed daily) added 4 hs prior to the PDE inhibitors, andincreasing concentrations of sildenafil or pentoxifylline. cGMP and cAMPlevels were measured in cell homogenates. Results are the means of 2separate experiments conducted in triplicate. (A) cGMP levels in thepresence of sildenafil. (B) cGMP levels in the presence ofpentoxifylline. (C) cAMP levels in the presence of pentoxifylline.

FIG. 15. Intensification by iNOS blockade of aging-related fibrosis inthe arterial media. Old male rats were given L-NIL for 3 weeks, or leftuntreated. Young untreated rats served as controls. Tissue sections wereobtained from penis (to visualize the dorsal and bulbo-urethral penilearteries) and from the aorta and femoral artery, and stained with Masson(SMC: red; collagen: blue). (A) Micrographs from selected arteries andtissue sections, as indicated. Bar=50 μm. (B) Quantitative imageanalysis (QIA) expressed as ratios of areas occupied by SMC andcollagen, as means+/−SEM. Aorta: A vs. B, C p<0.001; B vs. C p<0.01;Femoral: A vs B, C p<0.001; B vs. C p<0.01 Dorsal: A vs B, C P<0.001 Bvs. P<0.05; Bulbourethral; A vs B, C p<0.001; B vs. C: NS.

FIG. 16. Reduction by iNOS blockade of the aging-related stimulation ofthe nitrosative pathway in the media of the penile arteries. Sectionswere immunostained as indicated. Nitrotyrosine is a marker forperoxynitrite. (A) Micrographs from selected arteries and tissuesections, as indicated. Bar=50 μm. (B) QIA as on FIG. 15, expressed asintensity of immunostaining per area, as means+/−SEM. For iNOS: Dorsal:A vs. B p<0.05; A vs. C: N.S.; B vs C: p<0.05; Bulbo-urethral: A vs. B:p<0.05; A vs. C: N.S; B vs. C: p<0.05. For 3-nitro tyrosine: Dorsal: Avs. B: p<0.001; A vs. C: p<0.05; B vs. C: p<0.05; Bulbourethral: A vs. Bp<0.01; A vs. C: N.S; B vs. C: p<0.01.

FIG. 17. Intensification by iNOS blockade of aging-related oxidativestress in the arterial media. Tissue sections were immunostained forCu²⁺Zn²⁺ SOD and for Mn²⁺ SOD. (A) Micrographs from selected arteriesand tissue sections only for Cu⁺Zn²⁺ SOD, as indicated. Bar=50 μm. (B)QIA as on FIG. 16, expressed as intensity of immunostaining per area, asmeans+/−SEM. Dorsal; A vs C p<0.05; A vs. NS; B vs. NS; Bulbourethral; Avs. C P<0.001; A vs. B P<0.05; B vs. P<0.01 A vs B, C p<0.001; B vs. C:NS. Mn²⁺ SOD gave essentially similar results (not shown).

FIG. 18. Differential expression of another marker of oxidative stress,heme oxygenase 1, in the adventitia of the arterial wall. Sections wereimmunostained with an antibody against heme oxygenase I andcounterstained with hematoxylin. (A) Micrographs from the dorsal artery.Bar=50 μm. (B) QIA as on previous figures. Values expressed asmeans+/−SEM. *p<0.05: young vs old (t test).

FIG. 19. Reduction by iNOS blockade of the aging-related stimulation ofapoptosis in the media of the penile arteries. Sections wereimmunostained with the TUNEL procedure and counterstained with methylgreen. (A) Micrographs from selected arteries and tissue sections, asindicated. Bar=50 μm (B) QIA as on previous figures, expressed asapoptotic index (percent number of apoptotic cells/total number ofcells). as means+/−SEM. Dorsal: A vs. B, C p<0.001; B vs. C p<0.05;Bulbourethral: A vs. C p<0.001; A vs. p<0.05; B vs C p<0.01.

FIG. 20. Intensification by iNOS blockade of the aging-relatedstimulation of PAI expression in the media of the penile arteries.Sections were immunostained with an antibody against PAI andcounterstained with hematoxylin. (A) Micrographs from the dorsal artery.(B) QIA for both the dorsal penile and bulbourethral arteries, as onprevious figures. Bar=50 μm Values expressed as means+/−SEM. a vs. b,c:p<0.001.

FIG. 21. Effect of 8 Br-cGMP on apoptosis in fibroblasts from humancultured PD plaque, estimated by TUNEL. (A) Microphotographs (200×) ofapoptotic cells in incubations receiving no addition and 400 uM 8Br-cGMP for 3 days. (B) Apoptotic index, as means+/−SEM for threeseparate incubations. a vs b,c p>0.05.

Table 1. Differential profiles of selected gene expression in humanPeyronie's plaques and Dupuytren's nodules against their respectivecontrol tissues determined with a DNA microarray assay.

Table 2. Effect of aging on arterial wall thickness and lumen diameterin large and small arteries in the rat n=5; **: p<0.01.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Abbreviations andDefinitions

The following abbreviations are used herein. Other abbreviations notlisted below have their plain and ordinary meaning.

ASMA: α-smooth muscle actin;

ED: erectile dysfunction;

iNOS: inducible NOS (also NOS II);

L-NAME: L-N-Nitro-L-arginine methyl esther;

L-NIL: L-iminoethyl-L-lysine;

MMP: matrix metalloproteinase;

nNOS: neuronal NOS;

NO: nitric oxide;

NOS: nitric oxide synthase;

PAI: plasminogen activator inhibitor;

PD: Peyronie's disease;

PDE: phosphodiesterase;

PKG: protein kinase G;

PPC: pluripotent cells;

QIA: quantitative image analysis;

ROS: reactive oxygen species;

SMC: smooth muscle cell;

SNAP: S-Nitroso-N-acetyl penicillamine;

TA: tunica albuginea;

TGF-β1: transforming growth factor-β1;

TIMP: tissue inhibitor of MMP;

As used herein, “a” or “an” may mean one or more than one of an item.

This application concerns, at least in part, isolated proteins andnucleic acids for type 5 phosphodiesterase (PDE5, e.g., GenBankAccession Nos. NM033437, NM033431, NM033430, NM001083, NP246273,NP237223, NP236914, NP001074), as well as methods of therapeutictreatment of fibrotic diseases directed towards such proteins. In thepresent disclosure, reference to “PDE5” or “type 5 phosphodiesterase”without further qualification or limitation means any or all isoforms ofPDE5.

A “PDE5 isoform” is a variant of type 5 phosphodiesterase that differsin its primary structure (i.e., amino acid sequence) from other isoformsof PDE5. The term encompasses, but is not limited to, isoforms that areproduced by truncation, amino acid substitution (mutation) or byalternative mRNA splicing, so long as some difference in amino acidsequence results. For the purposes of the present invention, other typesof covalent modification would be considered to fall within the scope ofa single isoform. For example, both phosphorylated and unphosphorylatedforms of PDE5 would be considered to represent the same isoform.

As used herein, an “inhibitor” of PDE5 means any compound or combinationof compounds that acts to decrease the activity of PDE5, either directlyor indirectly. An inhibitor can be a molecule, an atom, or a combinationof molecules or atoms without limitation. The term “antagonist” of PDE5is generally synonymous with an “inhibitor” of PDE5. Inhibitors may actdirectly on PDE5 by, for example, binding to and blocking the catalyticsite or some other functional domain of PDE5 that is required foractivity. An inhibitor may also act indirectly, for example, byfacilitating or interfering with the binding of PDE5 to another proteinor peptide.

This application also concerns, at least in part, isolated proteins andnucleic acids for type 4 phosphodiesterase (PDE4, e.g., GenBankAccession Nos. NM006203, NM002600, NM006202, NP006194, NP002591,NP006193), as well as methods of therapeutic treatment of fibroticdiseases directed towards such proteins. In the present disclosure,reference to “PDE4” or “type 4 phosphodiesterase” without furtherqualification or limitation means any or all isoforms of PDE4. The terms“PDE4 isoform” and PDE4 “inhibitor” or “antagonist” are usedconsistently with the corresponding terms defined above for PDE5.

Peyronie's Disease and Other Fibrotic Conditions

Peyronie's Disease

Peyronie's disease (PD) is a localized fibrosis of the tunica albuginea(TA) of the penis (Hellstrom and Bivalacqua, 2000; Gonzalez-Cadavid etal., 2002; Gholami et al., 2002) affecting close to 5% of the malepopulation (Schwarzer et al., 2001). The leading theory of the etiologyof PD is that it results from an abnormal wound healing process of theTA subsequent to an injury, usually during coitus (Hellstrom andBivalacqua, 2000; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002;Jarow and Lowe, 1997; Devine et al., 1997). It is assumed that at thetime of the injury, extravasation of blood borne proteins, mainlyfibrin, into the TA occurs (Somers and Dawson, 1997; Van de Water, 1997;Herrick et al., 1999). Some of these foreign proteins may induce asevere but local inflammatory response in the TA resulting in the localrelease of pro-fibrotic factors, mainly transforming growth factor β1(TGF-β1) and reactive oxygen species (ROS) (Hellstrom and Bivalacqua,2000; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002), which thentrigger excessive deposition and disorganization of the collagen fibers.

Within the injured TA of some individuals, the above process may resultin: a) an increase in the differentiation of TA fibroblasts intomyofibroblasts (Vernet et al., 2002); b) an increased deposition ofcollagen fibers by both the TA fibroblasts and myofibroblasts (Vernet etal., 2002; Ferrini et al., 2002); c) a decrease in apoptosis of the TAfibroblasts/myofibroblasts, and d) a decrease in the natural breakdownand reorganization of newly deposited collagen fibers that is normallyperformed by the matrix metalloproteinases (MMP) (Mignatti et al., 1996;Arthur, 2000). The MMPs are the collagenolytic enzymes that are involvedin the natural turnover of collagen in the wound healing process. At itsextreme, this process may become excessive, with newly depositedcollagen and extracellular matrix in a tissue that fails to “heal andreorganize normally” (Mignatti et al., 1996) eventually becomingcalcified (Gelbard, 1988; Muralidhar et al., 1996). Calcification mayoccur by osteoblasts that are transformed from pluripotent cells (PPC)among the fibroblasts and/or myofibroblasts within the TA by eitherautocrine and/or paracrine factor(s).

The primary cell that is involved in collagen synthesis in the woundhealing process is the fibroblast (Singer and Clark, 1999). For woundclosure, some of the fibroblasts must differentiate into myofibroblasts(Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al.,2002; Muralidhar et al., 1996; Singer and Clark, 1999, Powel et al.,1999), cells that are intimately involved in the terminal stages of thewound healing process. Once this process is completed, the myofibroblastis normally eliminated from the wound by apoptosis (Gabbiani, 1996). Ifmyofibroblasts persist and do not undergo pre-programmed cell death,they may continue to synthesize additional collagen and extracellularmatrix, leading to an increase in fibrosis. Within the TA, this increasein fibrosis may lead to the clinical recognition of a palpablePeyronie's plaque.

In addition to being a stimulator of the differentiation of fibroblastsinto myofibroblasts, TGF-β1 can be secreted by both fibroblasts andmyofibroblasts (Powel et al., 1999; Tomasek et al., 1999; Walker et al.,2001). In other fibrotic conditions, like cardiac and renal fibrosis,TGF-β1 has been shown to not only increase the replication anddifferentiation of fibroblasts into myofibroblasts, but also to inhibitapoptosis of the myofibroblasts (Desmoulière, 1995; Chipev et al.,2000). This self-perpetuating cycle of cellular differentiation offibroblasts into myofibroblasts and continued TGF-β1 secretion by thesesame two cell types, as a result of the exposure of these cells toTGF-β1 itself, may ultimately lead to excessive collagen synthesis.Coupled with a disorganization of collagen fibers and a decrease intheir degradation by at least a partial inhibition of the MMPs (Iredale,1997), this may explain the continued growth of the PD plaque.

In response to the pro-fibrotic effects of TGF-β1 and ROS in the TA, theinjured TA tissue attempts to counteract these pro-fibrotic processes byreleasing the anti-fibrotic compound, nitric oxide (NO). NO in the TA issynthesized by the inducible nitric oxide synthase enzyme (iNOS) (Vernetet al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002;Ferrini et al., 2002). Therefore, in the development of PD, there may bea constant battle between pro-fibrotic and anti-fibrotic processeswithin the TA, as is evident from the results from DNA microarraysdiscussed in the Examples below, with the winner determining whethernormal healing or a fibrotic condition ensues.

As discussed in the Examples below, studies on the corresponding humantissues and cells combined with data from rat models have shown that thedevelopment of the PD plaque is associated with expression of induciblenitric oxide synthase (iNOS) and stimulation of nitric oxide (NO)synthesis, in conjunction with an increase in oxidative stress and ROSlevels (Vernet et al., 2002; Gholami et al., 2002; Hellstrom andBivalacqua, 2000; Sikka et al., 2002). The specific inhibition of iNOSactivity with L-iminoethyl-L-lysine (L-NIL) exacerbates fibrosis in theTGF-β1 rat model, consistent with a model wherein NO produced by iNOSplays an antifibrotic role in PD by at least three mechanisms: a) thequenching of the pro-fibrotic ROS by a reaction leading to the formationof peroxynitrite; b) the down-regulation of fibroblast replication andmyofibroblast differentiation; and c) the consequent or independentreduction in the transcriptional expression of collagen I.

An additional mechanism for NO to counteract fibrosis may involvestimulation of myofibroblast and/or fibroblast programmed cell death.The induction of apoptosis by NO is well documented, either in vitro byNO donors, such as S-Nitroso-N-acetyl penicillamine (SNAP) (Sikka etal., 2002; Nishio et al., 1996) or inducible nitric oxide synthase(iNOS) expression (Nishio et al., 1996; Tain et al., 2002), or in vivoby neuronal NOS (nNOS) activation (Ferrini et al., 2001b), iNOSinduction (Ferrini et al., 2001b; Watanabe et al., 2002), oradministration of the NOS substrate L-arginine (Wang et al., 1999; Holmet al., 2000). The proposed antifibrotic role of iNOS is in agreementwith indirect results obtained in animal models of kidney and cardiacfibrosis, where general NOS inhibitors (not isoform-specific), such asL-N_(ω)-Nitro-L-arginine methyl esther (L-NAME), cause or exacerbatefibrosis (Chatziantoniou et al., 1998; Boffa et al., 1999; Pechanova etal., 1999). L-arginine supplementation has been shown to beanti-fibrotic in vascular and renal disease (Peters et al., 2000), buthas not been tested on the PD plaque.

Thus, pharmacologic inhibition of the pro-fibrotic process and/orstimulation of the anti-fibrotic processes, may halt the progressionand/or reverse the process of PD. More globally, such results may beextrapolated to more life-threatening fibrotic conditions such as renal,lung, liver, and cardiac fibrosis (Nagase and Brew, 2002;Martinez-Hernández, 1994; Schuppan et al., 2000). The results disclosedherein provide novel avenues of therapy for not only PD but also forfibrosis in general.

The interaction within the TA between the pro-fibrotic and anti-fibroticfactors acting on fibroblasts and myofibroblasts and their respectivedifferentiation and apoptotic processes is outlined in FIG. 1. One ofthe major functions of NO produced by iNOS in the PD tissue is to reactwith the pro-fibrotic compound ROS to form peroxynitrite (Beckmann andKoppenol, 1996; Ferrini et al., 2001a; Vernet et al., 1998).Peroxynitrite is known to induce apoptosis in most cell types, andspecifically of the collagen-producing cells such as the fibroblast andmyofibroblast (Heigold et al., 2002; Duffield et al., 2000; Zhang andPhan, 1999). In addition, NO stimulates guanylyl cyclase to produce cGMPGonzalez-Cadavid et al., 1999), which in turn stimulates PKG (proteinkinase G) (Sinnaeve et al., 2002; Wollert et al., 2002). Like NO, bothcGMP and PKG inhibit collagen synthesis and are anti-fibrotic (Sinnaeveet al., 2002; Wollert et al., 2002; Hofmann et al., 2000; Chen et al.,1999a, 1999b; Redondo et al., 1998). cGMP is normally degraded toinactive GMP by the phosphodiesterase (PDE) enzymes (Corbin and Francis,1999; Uckert et al., 2001). Some of the inhibitors of these enzymes,like pentoxifylline, have also been shown to be anti-fibrotic in animalmodels and humans (Fischer et al., 2001; Desmouliere et al., 1999;Kremer et al., 1999).

The accumulation of collagen, which is one of the histological hallmarksof tissue fibrosis, may in part be also due to the inactivation of theMMP enzymes that degrade the already laid-down collagen fibers duringits natural turnover cycle (Mignatti et al., 1996; Arthur, 2000). MMPscan be inactivated by TIMPs, the tissue inhibitors of MMP, that havebeen shown to increase in fibrotic conditions (Iredale, 1997; McCruddenand Iredale, 2000; Arthur, 2000). Another anti-fibrotic effect of NO isthat it stimulates MMP activity (Sasaki et al., 1998; Okamoto et al.,1997) and inhibits the expression of TIMP (Darby et al., 2002; Bugno etal., 1999).

The results disclosed below provide a series of approaches (Magee etal., 2002b; Ferrini et al., 2002) focused on the role of themyofibroblast and the interaction between NO and ROS (the NO/ROSbalance) in the pathogenesis of the PD plaque and in arteriosclerosis,particularly of penile arteries. These include the use of theestablished TGF-β1 rat model of PD (Ferrini et al., 2002; Vernet et al.,2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002), theestablishment and study of cell cultures from the human PD and normal TAtissues (Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami etal., 2002), the application of quantitative image analysis (QIA) oftissue sections and cells subjected to histochemistry andimmunohistochemistry (Ferrini et al., 2002; Vernet et al., 2002;Gonzalez-Cadavid et al., 2002; Gholami et al., 2002), the use ofselective inhibitors of some of the biochemical pathways shown in FIG. 1(Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid et al.,2002; Gholami et al., 2002), DNA microarrays for multiple geneexpression (Magee et al., 2002b), the discovery that the same processesthat occur in the PD fibrotic plaque are also involved in aging-relatedfibrosis of the arterial wall media leading to arteriosclerosis, whichby affecting the penile arteries would cause ED, and other molecularbiology assays that provide an integrated analysis of the molecularpathophysiology of this condition, with corresponding novel approachesto therapeutic intervention of fibrotic disease directed towards theunderlying molecular pathways.

The combination of 1) an agent that increases NO, cGMP or cAMP levels,with 2) a compound that reduces oxidative stress and ROS levels, such asan antioxidant, will preserve the antifibrotic effects of agents in thefirst category, without an undesirable excessive level of apoptosis thatmay lead to cytotoxicity in cells other than myofibroblasts (e.g.,smooth muscle cells, neurons, endothelial cells, etc.) By reducing ROSwith the antioxidant, the formation of deleterious levels ofperoxynitrite (the product of ROS quenching by NO) would be reduced tothe minimum required for effective antifibrotic effects onmyofibroblasts and fibroblasts involved in excessive collagen andextracellular matrix synthesis, without damage to other tissues. In thecase of the combination of an antioxidant with agents in the firstcategory raising cGMP or cAMP levels, the reduction in ROS would allowmore endogenous NO levels to be preserved. Therefore, the combination ofagents may be more effective and safe than a single agent in eithercategory alone.

The skilled artisan will realize that the methods and compositionsdisclosed herein are of use not only for treatment of Peyronie's diseaseand ED due to loss of cavernosal smooth muscle in the trabecular spacesand penile arteries, but also for other conditions involving fibrosis,such as penile corporal fibrosis, Dupuytren's disease nodules, vaginalfibrosis, clitoral fibrosis, female sexual arousal disorder, abnormalwound healing, keloid formation, general fibrosis of the kidney,bladder, prostate, skin, liver, lung, heart, intestines or any otherlocalized or generalized fibrotic condition, vascular fibrosis, arterialintima hyperplasia, atherosclerosis, arteriosclerosis, restenosis,cardiac hypertrophy or any other condition characterized by excessivefibroblast or smooth muscle cell proliferation or deposition of collagenand extracellular matrix in the blood vessels and/or heart. Both thevagina and clitoris are known to undergo fibrosis and hardening withaging, menopause and estrogen/testosterone deficiency. Together withpoor lubrication, the vaginal/clitoral fibrosis contribute to thedevelopment of female sexual arousal disorder, affecting about 30 to 40%of women. (Traish et al., Arch. Sex Behav. 31:393-400, 2002; Park etal., Intl. J. Impot. Res. 13:116-124, 2001; Berman et al., J. SexMarital Ther. 27:411-420, 2001; Berman et al., Urology 54:385-391, 1999;Berman et al., Fertil. Steril., 79:925-931, 2003.) The mechanisms offibrosis are similar for a number of different organs and diseasestates.

A distinction exists between long-term (weeks, months, years) continuoustreatment with, for example, a PDE5 inhibitor such as sildenafil tomaintain a constant level of these agents in order to arrest or regressa fibrotic condition, versus on demand (prior to the sexual act) singlepill, short-term treatment with sildenafil or other PDE5 inhibitors toobtain smooth muscle vasodilation in the penis (male penile erection) orvagina/clitoris (female sexual arousal) upon sexual stimulation. Currentstudies with sildenafil are symptomatic to treat defects invaginal/clitoral or penile vasodilation exclusively during a sexual actand are not addressed to the long-term cure of underlying tissuefibrosis. Additional details relevant to the treatment of fibroticconditions are disclosed in the Examples section below as well as in thereferences of Vernet et al. (2002), Gonzalez-Cadavid et al. (2002) andGholami et al. (2002), the entire texts of which are specificallyincorporated herein by reference.

Peripheral Vascular Disease, Erectile Disfunction and Hypertension

One of the prevalent views of peripheral vascular disease is that it iscaused by oxidative damage to the arterial wall by reactive oxygenspecies (ROS), that cause lipid peroxidation and other alterations (Caiand Harrison, 2000; Berry et al., 2001; Zalba et al., 2000). Thesecompounds are mainly produced by xanthine oxidase, NADPH oxidase, aswell as mitochondrial enzymes, and are counteracted by heme-oxygenase Iand superoxide dismutase (SOD), which can reduce ROS by acting asendogenous antioxidants. In addition to causing endothelial damage, ROSare known stimulators of collagen deposition and SMC proliferation(Berry et al., 2001; Zalba et al., 2000) in the vascular wall. Xanthineoxidase and SOD are also present in the penile corpora cavernosa (Joneset al., 2002), and oxidative stress due to ROS has been postulated to becentral to impaired cavernosal function in aging-related ED (Jones etal., 2002; Khan et al., 2001; Bivalacqua et al., 2003).

Besides antioxidants, nitric oxide (NO) also quenches ROS in thevasculature, as shown by the increase in ROS levels and the developmentof cardiac and renal fibrosis and vascular stiffness when there islong-term systemic blockade of NOS activity with NOS inhibitors(Kitamoto et al., 2000; Gonzalez et al, 2000; Usui et al., 1999). TheROS-quenching and anti-fibrotic effects of NO are not limited to the SMCand can be demonstrated in other non-vascular conditions (Ferrini etal., 2002; Vernet et al., 2002). In this process, NO reduces ROS levelsthrough the formation of peroxynitrite (Cai and Harrison, 2000; Jones etal., 2002; Ferrini et al., 2002; Vernet et al., 2002; Gewaltig andKojda, 2002), thereby increasing the NO/ROS ratio. NO is also postulatedto not only inhibit collagen synthesis directly, but to favor collagendegradation by stimulating metalloproteinases and down-regulatingexpression of their inhibitors, such as the plasminogen activatorinhibitor (PAI) (Li et al., 2000; Kaikita et al., 2002). Thepredominance of nitrosative pathways over oxidative stress is proposedto be protective against fibrosis (Ferrini et al., 2002; Vernet et al.,2002), ED (Jones et al., 2002), atherosclerosis, and hypertension(Gewaltig and Kojda, 2002; Cheng et al., 2001).

The NO/ROS balance also directly modulates the relaxation of thevascular and penile smooth muscle. The NO produced by the endothelialNOS in the vascular endothelium controls blood pressure by relaxing thearterial SMC (González-Cadavid et al., 1999). In the penile corporacavernosa, NO as a mediator of penile erection is produced by theneuronal NOS, specifically the PnNOS variant (Berry et al., 2001),localized in the nerve terminals, and to a lesser extent by endothelialNOS in the lacunar and sinusoidal endothelium of the penis(González-Cadavid et al., 1999). In experimental animals, reduction inNOS levels in the vasculature and penile corpora is associated withhypertension (Gewaltig et al., 2002) and with ED, respectively(González-Cadavid et al., 1999; Garban et al., 1995; Berry et al.,2001). If oxidative stress becomes excessive, the reaction of ROS withNO to form peroxynitrite reduces NO concentration in the tissues, whichmay lead to hypertension and ED by impairing NO dependent SMCrelaxation.

It is still unknown to what extent these neuronal and endothelial NOSisoforms participate in producing NO as an antifibrotic mechanism. Incontrast, more direct evidence has emerged recently on the role of theinducible isoform of NOS (iNOS) (Kibbe et al., 1999) in reducing ROS andmodulating the SMC/collagen ratio in different tissues. iNOS isspontaneously induced in the corpora cavernosa (Ferrini et al., 2001a)and brain (Vernet et al., 1998; Ferrini et al., 2001b) during aging, andin certain fibrotic conditions (Ferrini et al., 2002; Vernet et al.,2002). In the vasculature, iNOS is also induced in the media inaging-related arterial stiffness (Goettsch et al., 2001; Chou et al.,1998; Cernadas et al., 1998), transplant arteriosclerosis (Lee et al.,1999), and atherosclerosis (Ihrig et al., 2001; Niu et al., 2001;Behr-Roussel et al., 2000), and it is assumed to inhibit collagendeposition and prevent medial hyperplasia via induction of SMC apoptosisand/or inhibition of SMC replication (Gewaltig and Kojda, 2002; Kibbe etal., 1999; Niu et al., 2001). The specific inhibition of iNOS activityby L-N-(iminocthyl)-lysine acetate (L-NIL) (Ferrini et al., 2002; Vernetet al., 2002; Behr-Roussel et al., 2000), or the blockade of iNOSexpression in the iNOS knockout mouse (Ihrig et al., 2001; Niu et al.,2001; Hochberg et al., 2000), causes fibrosis in non-vascular tissues, adecrease in NO/peroxynitrite levels, an increase in ROS, and a reductionin the SMC/collagen ratio.

Despite the fact that a certain predominance of the nitrosative over theoxidative pathways may preserve the normal integrity and function ofblood vessels and corpora cavernosa, an excessive production of NO andperoxynitrite, may also induce apoptosis and cell loss (Ferrini et al.,2001a, 2002; Vernet et al., 2002; Kibbe et al., 1999). Depending on thecontext, this may be beneficial by preventing media hyperplasia inatherosclerosis and restenosis and ameliorate fibrosis in other systems(Ferrini et al., 2002; Vernet et al., 2002; Gewaltig et al., 2002;Behr-Roussel et al., 2000). But excessive peroxynitrite may also benoxious, if it leads to a loss of SMC and the subsequent impairment oftissue relaxation. We propose that during aging, iNOS induction in thevasculature is not restricted to the cavernosal SMC (Ferrini et al.,2001a) and large arteries (Goettsch et al., 2001; Chou et al., 1998;Cernadas et al., 1998), but is generalized to the wall of the entireperipheral vascular tree. This process would aim to counteract oxidativestress and metalloproteinase inhibition, and the subsequent decrease inthe SMC/collagen ratio that causes loss of compliance and NO-inducedvaso-relaxation. As disclosed in the following Examples, we haveexamined large and small (resistance) arteries in both young and agedrats for SMC/collagen ratio, iNOS, peroxynitrite, heme oxygenase I, SOD,PAI, and SMC apoptosis, and determined how these parameters wereaffected in aged rats when iNOS activity was specifically inhibited withL-NIL.

NO/cGMP Inhibition of Fibrogenic Pathways

In molecular terms, the fibrotic process occurring during abnormal woundhealing, e.g., in dermal wounds, is essentially an increased anddisorganized collagen deposition impairing granulation tissue formation.This is accompanied by an increase in the local production and secretionof TGF-(1 (Klar and Morrisey, 1998; Badalamente et al., 1996; Wahl,1997), a factor which: a) stimulates collagen synthesis (Tiggelman etal., 1997; Faouzi et al., 1999), b) inhibits collagenolysis (van der Zeeet al., 1997) and fibrinolysis (Holmdahl et al., 2001), c) enhances therelease of ROS (Casini et al., 1997; Muriel, 1998a), and d)transcriptionally represses iNOS (Hung et al., 1995).

ROS are hydroxyl radicals and superoxide anions that are quenched by NOto primarily form peroxynitrite (Poli, 2000; Curtin et al., 2002;Cattell, 2002; Kim et al., 2001; Fan et al., 2000; Ito et al., 1992).The balance between NO and ROS levels is known to be considerablyaltered in other fibrotic conditions affecting liver (cirrhosis), lung(pulmonary fibrosis), kidney (renal fibrosis), heart (cardiachypertrophy), and the vascular tree (arterial medial hyperplasia). Thisabnormal ratio between NO and ROS is believed to be due to both adecrease in local NO synthesis (presumably via iNOS) and an increase inROS (Casini et al., 1997; Muriel, 1998a; Curtin et al., 2002; Cattell,2002; Kim et al., 2001; Fan et al., 2000). ROS, produced by themacrophages and neutrophils, have been shown to induce lipidperoxidation in cell membranes and increase vascular permeability andleakage of fibrinogen and other clotting factors into tissue (Cattell,2002; Kim et al., 2001). ROS generation during oxidative stress isaccompanied by a considerable induction of heme-oxygenase-1 (HO-1)(Foresti et al., 1999; Nathan, 1997), the enzyme that protects againstoxidative stress and acts as an anti-apoptotic and anti-inflammatory(Ryter and Choi, 2002) response. HO-1 can also be elicited byperoxynitrite.

Among the several regulators of collagen deposition and wound healing,NO is particularly interesting as an inhibitor of fibrosis. In the caseof PD, NO appears to be produced by the induction of iNOS in the TA(Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid et al.,2002; Gholami et al., 2002). This iNOS isoform is involved in producingpersistent high levels of NO by transcriptional induction, essentiallyas a defensive mechanism during inflammation (Nathan, 1997a, 1997b).iNOS is physiologically expressed in the adult at very low basal levels,if at all. Only upon induction by cytokines, such as tumor necrosisfactor α (TNFα), interleukin 1β (IL-1β), interferon-γ (INFγ), andrelated factors, does iNOS induction take place. It can under certainchronic conditions lead to a high, and some times excessive productionof NO that acts as either a cytotoxic agent, or, in the specific case ofcollagen, inhibits fiber deposition. These conditions includeinflammation, infections, cancer, degenerative diseases and aging, wherethe factors triggering this increased iNOS response are unknown (Kibbeet al., 1999; Wang et al., 2002a; Miller and Sandoval, 1999).Additionally, many NO metabolites, particularly peroxynitrite, triggerlocalized apoptosis and tissue toxicity (Nathan, 1997a).

The specific role of NO as a regulator of wound healing is wellestablished in vivo and in vitro (Curtin et al., 2002; Cattell, 2002;kim et al., 2001; Hogaboam et al., 1998; Rizvi and Myers, 1997; Cao etal., 1997; Chatziantoniou et al., 1998; Kolpakov et al., 1995). NOdonors and the NOS substrate, L-arginine, have been shown to inhibitcollagen fiber ((Curtin et al., 2002; Cattell, 2002; kim et al., 2001;Hogaboam et al., 1998; Rizvi and Myers, 1997; Cao et al., 1997;Chatziantoniou et al., 1998; Kolpakov et al., 1995) and fibrindeposition (Westenfeld et al., 2002; Dambisya and Lee, 1996; Catani etal., 1998; Dambisya et al., 1996), and TGF-β1 synthesis (Craven et al.,1997). The experimental decrease of NO synthesis by NOS inhibition, orthe reduction of iNOS induction, leads to impaired wound healing(Schaffer et al., 1997a), and also to fibrosis, as documented inmyocardial hypertrophy, coronary vascular remodeling following aninfarct, cystic fibrosis, obstructive nephropathy, and pulmonaryfibrosis (Takemoto et al., 1997; babal et al., 1997; Numaguchi et al.,1995; Ikeda et al., 1997; Moreno et al., 1996; Morrissey et al., 1996;Kelley and Drumm, 1998). Physiologically, the reduction of NO synthesismay occur by either transcriptional blockade of iNOS induction (Gellerand Billiar, 1998; Forstermann et al., 1998), and in certain cases,down-regulation of eNOS (Forstermann et al., 1998), or by inhibition ofNOS activity by advanced glycation-end products (AGE) (Jiaan et al.,1995) or a natural NOS competitive inhibitor such as asymmetric dimethylarginine (ADMA) (Boger et al., 1998).

In contrast to the anti-fibrotic effect of NO that would occur as adefense against fibrosis, in the early stages of normal wound healing NOactually stimulates collagen synthesis (Schaffer et al., 1997b; Yamasakiet al., 1998; Thornton et al., 1998; Sherratt and Dallon, 2002;Diegelmann, 1997). Therefore, the anti-fibrotic effects of NO may be theresult of a continuous and high level of local NO synthesis, like theone produced upon iNOS induction (Ferrini et al., 2002; Vernet et al.,2002). This shows the importance of the local levels of NO for eitherfacilitating normal collagen deposition (wound healing) or preventingits accumulation (fibrosis).

Anti-fibrotic effects of NO may also be mediated by cGMP throughguanylyl cyclase activation (Gonzalez-Cadavid et al., 1999). cGMPanalogs inhibit collagen synthesis (Chen et al., 1999a, 1999b; Redondoet al., 1998), fibroblast replication (Chiche et al., 1998; Pandey etal., 2000), myofibroblast differentiation (Tao et al., 1999), andpromote apoptosis (Loweth et al., 1997; Sirotkin et al., 2000; Taimor etal., 2000). PDE inhibitors, by elevating cGMP, also cause similareffects in vitro (Schade et al., 2002; Horio et al., 1999; Thompson etal., 2000), and in particular induce apoptosis in vivo (Chan et al.,2002; Takuma et al., 2001). Some of these PDE inhibitors, likepentoxifylline, are active in preventing experimental fibrosis in thelung, liver, and heart (Fischer et al., 2001; Desmouliere et al., 1999;Kremer et al., 1999), and are currently being used for the treatment ofhuman liver fibrosis (Windmeier and Gressner, 1997) and Crohn's disease(Reimund et al., 1997).

Role of Myofibroblast in Fibrosis

One of the major pathological findings in tissue fibrosis is thepresence of activated and proliferating myofibroblasts. These cells notonly play an important role in the contraction phase of normal woundhealing but they also are responsible for the development of tissuefibrosis or of a scar (Powel et al., 1999; Tomasek et al., 1999; Walkeret al., 2001). The myofibroblast (FIG. 1) is the cell widely believed togenerate the contracture in Dupuytren's disease, the condition presentin 10-20% of PD cases (Connelly, 1999). It is believed that afterfulfilling its role in wound contraction and in secreting collagenduring healing, the myofibroblast disappears by programmed cell death(apoptosis) (Gabbiani, 1996). However, in Dupuytren's disease and otherfibrotic conditions the myofibroblast persists, resulting in apersistent fibrosis and contracture (Kloen, 1999; Badalamente and Hurst,1999). Morphologically, myofibroblasts are intermediate between thefibroblast and the smooth muscle cell. Phenotypically, they expresslarge bundles of actin filaments (actin, myosin, and associatedproteins: “stress fibers”), with a fibrillar space material named the“fibronexus”, composed of fibronectin. Myofibroblasts can be identifiedby the detection of both a smooth muscle actin (ASMA) (absent infibroblasts), and vimentin (absent in smooth muscle cells).

It is believed that myofibroblasts can originate from eitherfibroblasts, smooth muscle cells, or from an as yet uncharacterized stemcell (Powel et al., 1999; Tomasek et al., 1999; Walker et al., 2001).Upon the appropriate stimulation, the myofibroblast may revert back to afibroblast or smooth muscle cell. Myofibroblasts express receptors forTGF-β1, PDGF, bFGF, endothelin, and prostaglandins. All these factorsgenerate in culture, an “activated” myofibroblast that is able toproliferate. In liver fibrosis, this activated form of the myofibroblastcan be transformed into the non-proliferating “stellate” form by eithercAMP or PGE2 (Powel et al., 1999; Tomasek et al., 1999; Walker et al.,2001; Wu and Zern, 2000). The activated myofibroblast is then able tosecrete cytokines, TGF-β1 and other growth factors, and inflammatorymediators. Within the latter category, the cell has been shown torelease NO and ROS, and matrix proteins involved in wound repair andfibrosis, such as collagens I, III, IV, VI, and XVIII, laminins,proteoglycans, adhesion molecules and MMPs (Powel et al., 1999; Tomaseket al., 1999; Walker et al., 2001).

The myofibroblast is involved in functions such as wound repair in skinand repair of the myocardium after myocardial infarction. This cell hasbeen implicated in the pathophysiology of the Dupuytren contracture,keloids, myocardial fibrosis, ischemia reperfusion injury, coronaryartery restenosis, glomerulonephritis, liver cirrhosis, pulmonaryinterstitial fibrosis, and many other fibrotic conditions (Powel et al.,1999; Tomasek et al., 1999; Walker et al., 2001). However, in PD, thereare few reports on the role of the myofibroblasts, other than thedescription of the original culture of fibroblasts from the PD plaque(Somers et al., 1982) and a few more recent studies (Anderson et al.,2000a, 2000b; Mulhall et al., 2001a, 2001b) and our own work (Vernet etal., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002). In cellculture, PD fibroblasts demonstrate a faster replication rate ascompared to those from the normal TA, a higher production of apro-fibrotic agent (basic fibroblast growth factor), and a potentialalteration of the p53 pathway that normally represses cell replicationand favors apoptosis, which would indicate a sort of “immortalization”in culture (Anderson et al., 2000a, 2000b; Mulhall et al., 2001a,2001b). Other groups have studied fibroblast cultures from thePeyronie's plaque but did not focus on their myofibroblast content(Duncan et al., 1991; El-Sakka et al., 1997a).

Animal Models of PD

An animal model of PD has recently been developed (El-Sakka et al.,1997b; El-Sakka et al., 1998), based on the administration of asynthetic heptapeptide of human TGF-β1 directly into the TA of the rat.After 45 days, the animal develops histological alterations and collagendeposition resembling those observed in the human PD plaque (El-Sakka etal., 1997a, 1997b, 1998). The administration of the full human TGF-β1protein to the TA leads to a similar process in the rat model (Vernet etal., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002; El-Sakkaet al., 1999, 1998).

Another potentially useful animal model for the study of thepathophysiology of the PD plaque is the collagen I promoter transgenicmouse (Fakhouri et al., 2001; Tharaux et al., 2000). This mouse carriesthe regulatory region of the collagen I-α2 gene linked to two reportergenes, luciferase and β-galactosidase, so that whenever collagen mRNAsynthesis is stimulated luciferase and β-galactosidase will beexpressed. Both proteins can be estimated by a chemiluminescencereaction in tissue homogenates, and β-galactosidase specifically by thedevelopment of a blue color in tissue sections.

This collagen 1 promoter mouse model has recently been used (Dussaule etal., 2000) to demonstrate the link between NO, endothelin, and collagensynthesis in kidney fibrosis, which is characterized by collagen Iaccumulation. In essence, by giving the NOS inhibitor L-NAME to thesetransgenic mice for up to 14 weeks, it was possible to inducenephroangio- and glomerulo-fibrosis, accompanied by an increase inluciferase levels and an increased urinary excretion rate of endothelin.The blockade of endothelin receptors with the selective ET antagonistbosentan reduced collagen deposition in the L-NAME animals, andabolished collagen I promoter activation, as quantitated by luciferaseactivity. This animal model demonstrated that NO inhibition induces anearly activation of the collagen I gene in the kidney arterioles andglomeruli, suggesting that NO inhibits collagen deposition and theendothelin-mediated fibrogenic effect, as confirmed by other studies(Boffa et al., 1999; Tharaux et al., 1999).

Nucleic Acids

In certain embodiments of the present invention, genes encoding one ormore isoforms of PDE4, PDE5, PKG, NOS, MMP or another protein may beincorporated into expression vectors for therapeutic use in fibrosis. Asdiscussed below, a gene encoding a given protein may contain a varietyof different bases and yet still produce a corresponding polypeptidethat is indistinguishable functionally, and in some cases structurally,from the known sequences of such genes. It is a matter of routine forthe skilled artisan to obtain known genomic and/or cDNA sequencesencoding various proteins from publicly available sources, such asGenBank.

Any reference to a nucleic acid should be read as encompassing a hostcell containing that nucleic acid and, in some cases, capable ofexpressing the product of that nucleic acid. Cells expressing nucleicacids of the present invention may prove useful in the context ofscreening for agents that induce, repress, inhibit, augment, interferewith, block, abrogate, stimulate, or enhance the catalytic activityand/or regulatory properties of PDE4 and/or PDE5.

Nucleic acids according to the present invention may contain an entiregene, a cDNA, or a domain of a protein that expresses catalyticactivity. The nucleic acid may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA).

The DNA segments of the present invention include those encodingbiologically functional equivalent proteins and peptides. Such sequencesmay arise as a consequence of codon redundancy and amino acid functionalequivalency that are known to occur naturally within nucleic acidsequences and the proteins thus encoded. Alternatively, functionallyequivalent proteins or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein structuremay be engineered, based on considerations of the properties of theamino acids being exchanged. Changes designed by man may be introducedthrough the application of site-directed mutagenesis techniques or maybe introduced randomly and screened later for the desired function, asdescribed below.

Expression Vectors

Nucleic acids encoding proteins or peptides may be incorporated intoexpression vectors for production of the encoded proteins or peptides.Non-limiting examples of expression systems known in the art includebacteria such as E. coli, yeast such as Pichia pastoris, baculovirus,and mammalian expression systems such as in COS or CHO cells. A completegene can be expressed or, alternatively, fragments of the gene encodingportions of polypeptide can be produced.

The gene or gene fragment encoding a polypeptide may be inserted into anexpression vector by standard subcloning techniques. An E. coliexpression vector may be used which produces the recombinant polypeptideas a fusion protein, allowing rapid affinity purification of theprotein. Examples of such fusion protein expression systems are theglutathione S-transferase system (Pharmacia, Piscataway, N.J.), themaltose binding protein system (NEB, Beverley, Mass.), the FLAG system(IBI, New Haven, Conn.), and the 6×His system (Qiagen, Chatsworth,Calif.).

Some of these systems produce recombinant polypeptides bearing only asmall number of additional amino acids, which are unlikely to affect theantigenic ability of the recombinant polypeptide. For example, both theFLAG system and the 6×His system add only short sequences, both of whichare known to be poorly antigenic and which do not adversely affectfolding of the polypeptide to its native conformation. Other fusionsystems are designed to produce fusions wherein the fusion partner iseasily excised from the desired polypeptide. In one embodiment, thefusion partner is linked to the recombinant polypeptide by a peptidesequence containing a specific recognition sequence for a protease.Examples of suitable sequences are those recognized by the Tobacco EtchVirus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (NewEngland Biolabs, Beverley, Mass.).

The expression system used may also be one driven by the baculoviruspolyhedron promoter. The gene encoding the polypeptide may bemanipulated by standard techniques in order to facilitate cloning intothe baculovirus vector. One baculovirus vector is the pBlueBac vector(Invitrogen, Sorrento, Calif.). The vector carrying the gene for thepolypeptide is transfected into Spodoptera frugiperda (Sf9) cells bystandard protocols, and the cells are cultured and processed to producethe recombinant antigen. See U.S. Pat. No. 4,215,051 (incorporated byreference).

Amino acid sequence variants of the polypeptide may also be prepared.These may, for instance, be minor sequence variants of the polypeptidewhich arise due to natural variation within the population or they maybe homologues found in other species. They also may be sequences whichdo not occur naturally but which are sufficiently similar that theyfunction similarly and/or elicit an immune response that cross-reactswith natural forms of the polypeptide. Sequence variants may be preparedby standard methods of site-directed mutagenesis such as those describedherein.

Substitutional variants typically contain an alternative amino acid atone or more sites within the protein, and may be designed to modulateone or more properties of the polypeptide such as stability againstproteolytic cleavage. Substitutions preferably are conservative, thatis, one amino acid is replaced with one of similar size and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: arginine to lysine; asparagine to glutamine orhistidine; aspartate to glutamate; cysteine to serine; glutamine toasparagine; glutamate to aspartate; histidine to asparagine orglutamine; isoleucine to leucine or valine; leucine to valine orisoleucine; lysine to arginine or glutamine; methionine to leucine orisoleucine; phenylalanine to tyrosine, leucine or methionine; serine tothreonine; threonine to serine; tryptophan to tyrosine; tyrosine totryptophan or phenylalanine; and valine to isoleucine or leucine.

Insertional variants include fusion proteins such as those used to allowrapid purification of the polypeptide and also may include hybridproteins containing sequences from other proteins and polypeptides whichare homologues of the polypeptide. For example, an insertional variantmay include portions of the amino acid sequence of the polypeptide fromone species, together with portions of the homologous polypeptide fromanother species. Other insertional variants may include those in whichadditional amino acids are introduced within the coding sequence of thepolypeptide. These typically are smaller insertions than the fusionproteins described above and are introduced, for example, to disrupt aprotease cleavage site.

The engineering of DNA segment(s) for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. It is believed that virtuallyany expression system may be employed in the expression of the claimednucleic acid sequences.

As used herein, the terms “engineered” and “recombinant” cells areintended to refer to a cell into which an exogenous DNA segment or gene,such as a cDNA or gene has been introduced through the hand of man.Therefore, engineered cells are distinguishable from naturally occurringcells that do not contain a recombinantly introduced exogenous DNAsegment or gene. Recombinant cells include those having an introducedcDNA or genomic gene, and also include genes positioned adjacent to aheterologous promoter not naturally associated with the particularintroduced gene.

To express a recombinant encoded protein or peptide, whether mutant orwild-type, in accordance with the present invention one would prepare anexpression vector that comprises one of the claimed isolated nucleicacids under the control of, or operatively linked to, one or morepromoters. To bring a coding sequence “under the control of” a promoter,one positions the 5′ end of the transcription initiation site of thetranscriptional reading frame generally between about 1 and about 50nucleotides “downstream” (i.e., 3′) of the chosen promoter. The“upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded recombinant protein. This is the meaning of“recombinant expression” in this context.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein or peptide expression in a variety of host-expression systems.Cell types available for expression include, but are not limited to,bacteria, such as E. coli and B. subtilis transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.

Promoters that are most commonly used in recombinant DNA constructioninclude the g-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enablingthose of skill in the art to ligate them functionally with plasmidvectors.

For expression in Saccharomyces, the plasmid YRp7, for example, iscommonly used (Stinchcomb et al., Nature, 282: 39, 1979; Tschemper etal., Gene, 10:157, 1980). This plasmid contains the trpl gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. Thepresence of the trpl lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073,1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.,7:149, 1968; Holland et al., Biochemistry, 17:4900, 1978), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other suitable promoters, which have the additional advantage oftranscription controlled by growth conditions, include the promoterregion for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization.

In addition to micro-organisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing one or more coding sequences.

Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2,3T3, RIN and MDCK cell lines. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and processes the gene product in the specific fashion desired.Such modifications (e.g., glycosylation) and processing (e.g., cleavage)of protein products may be important for the function of the encodedprotein. In preferred embodiments of the invention, the host cells arehuman cells inside a subject with a fibrotic condition.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecells lines or host systems may be chosen to ensure the correctmodification and processing of the foreign protein expressed. Expressionvectors for use in mammalian cells ordinarily include an origin ofreplication (as necessary), a promoter located in front of the gene tobe expressed, along with any necessary ribosome binding sites, RNAsplice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Further, itis also possible, and may be desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2, and mostfrequently Simian Virus 40 (SV40). The early and late promoters of SV40virus are particularly useful because both are obtained easily from thevirus as a fragment that also contains the SV40 viral origin ofreplication. Smaller or larger SV40 fragments may also be used, providedthere is included the approximately 250 bp sequence extending from theHind site toward the Bgl I site located in the viral origin ofreplication.

In cases where an adenovirus is used as an expression vector, the codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing proteins in infectedhosts.

Specific initiation signals may also be required for efficienttranslation of the claimed isolated nucleic acid coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Exogenous translational control signals, including the ATG initiationcodon, may additionally need to be provided. One of ordinary skill inthe art would readily be capable of determining this and providing thenecessary signals. It is well known that the initiation codon must bein-frame (or in-phase) with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons may be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements or transcription terminators (Bittner et al., Methods inEnzymol, 153: 516-544, 1987).

In eukaryotic expression, one will also typically desire to incorporateinto the transcriptional unit an appropriate polyadenylation site (e.g.,5′-AATAAA-3′) if one was not contained within the original clonedsegment. Typically, the poly A addition site is placed about 30 to 2000nucleotides “downstream” of the termination site of the protein at aposition prior to transcription termination.

Nucleic Acid Delivery

Liposomal Formulations

In certain broad embodiments of the invention, the oligo- orpolynucleotides and/or expression vectors may be entrapped in aliposome. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self-rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, In: LiverDiseases, Targeted Diagnosis and Therapy Using Specific Receptors andLigands, Wu et al. (Eds.), Marcel Dekker, New York, pp 87-104, 1991).Also contemplated are cationic lipid-nucleic acid complexes, such aslipofectamine-nucleic acid complexes.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989).In other embodiments, the liposome may be complexed or employed inconjunction with nuclear non-histone chromosomal proteins (HMG-1). Inthat such expression vectors have been employed in transfer andexpression of a polynucleotide in vitro and in vivo, they may beapplicable for the present invention. Liposomes within the scope of thepresent invention can be prepared in accordance with known laboratorytechniques. In one embodiment, liposomes are prepared by mixingliposomal lipids, in a solvent in a container, e.g., a glass,pear-shaped flask. The container should have a volume ten-times greaterthan the volume of the expected suspension of liposomes. Using a rotaryevaporator, the solvent is removed at approximately 40° C. undernegative pressure. The solvent normally is removed within about 5 min to2 hours, depending on the desired volume of the liposomes. Thecomposition can be dried further in a desiccator under vacuum. The driedlipids generally are discarded after about 1 week because of a tendencyto deteriorate with time.

The dried lipids or lyophilized liposomes prepared as described abovemay be reconstituted in a solution of nucleic acid and diluted to anappropriate concentration with a suitable solvent. The mixture is thenvigorously shaken in a vortex mixer. Unencapsulated nucleic acid isremoved by centrifugation at 29,000′g and the liposomal pellets washed.The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount of nucleicacid encapsulated can be determined in accordance with standard methods.After determination of the amount of nucleic acid encapsulated in theliposome preparation, the liposomes may be diluted to appropriateconcentration and stored at 4° C. until use.

Alternative Delivery Systems

Adenoviruses:

Human adenoviruses are double-stranded DNA tumor viruses with genomesizes of approximate 36 kB. As a model system for eukaryotic geneexpression, adenoviruses have been widely studied and wellcharacterized, which makes them an attractive system for development ofadenovirus as a gene transfer system. This group of viruses is easy togrow and manipulate, and they exhibit a broad host range in vitro and invivo. In lytically infected cells, adenoviruses are capable of shuttingoff host protein synthesis, directing cellular machineries to synthesizelarge quantities of viral proteins, and producing copious amounts ofvirus.

The E1 region of the genome includes E1A and E1B, which encode proteinsresponsible for transcription regulation of the viral genome, as well asa few cellular genes. E2 expression, including E2A and E2B, allowssynthesis of viral replicative functions, e.g. DNA-binding protein, DNApolymerase, and a terminal protein that primes replication. E3 geneproducts prevent cytolysis by cytotoxic T cells and tumor necrosisfactor and appear to be important for viral propagation. Functionsassociated with the E4 proteins include DNA replication, late geneexpression, and host cell shutoff. The late gene products include mostof the virion capsid proteins, and these are expressed only after mostof the processing of a single primary transcript from the major latepromoter has occurred. The major late promoter (MLP) exhibits highefficiency during the late phase of the infection (Stratford-Perricaudetand Perricaudet, In: Human Gene Transfer, O. Cohen-Haguenauer et al.,eds., John Libbey Eurotext, France, pp. 51-61, 1991).

As only a small portion of the viral genome appears to be required incis (Tooze, Molecular Biology of DNA Tumor Viruses, 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1991), adenovirus-derivedvectors offer excellent potential for the substitution of large DNAfragments when used in connection with cell lines such as 293 cells.Ad5-transformed human embryonic kidney cell lines (Graham et al., J.Gen. Virol., 36:59-72, 1977) have been developed to provide theessential viral proteins in trans.

Advantages of adenovirus vectors over retroviruses include the higherlevels of gene expression. Adenovirus replication is independent of hostgene replication, unlike retroviral sequences. Because adenovirustransforming genes in the E1 region can be readily deleted and stillprovide efficient expression vectors, oncogenic risk from adenovirusvectors is thought to be low (Grunhaus and Horwitz, Seminar in Virology,3:237-252, 1992).

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus which is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Sequences encoding relatively largeforeign proteins can be expressed when additional deletions are made inthe adenovirus genome. For example, adenoviruses deleted in both E1 andE3 regions are capable of carrying up to 10 kB of foreign DNA and can begrown to high titers in 293 cells (Stratford-Perricaudet andPerricaudet, 1991). Persistent expression of transgenes followingadenoviral infection has also been reported.

Other Viral Vectors as Expression Constructs.

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York,Plenum Press, pp. 117-148, 1986) adeno-associated virus (AAV) (Baichwaland Sugden, 1986) and herpes viruses may be employed. They offer severalattractive features for various mammalian cells (Horwich, et al., J.Virol., 64:642-650, 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al.,Hepatology, 14:124A, 1991).

Non-Viral Methods.

Several non-viral methods for the transfer of expression vectors intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and van der Eb,Virology, 52:456-467, 1973) DEAE-dextran (Gopal, Mol. Cell Biol.,5:1188-1190, 1985), lipofectamine-DNA complexes, and receptor-mediatedtransfection (Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, J.Biol. Chenm., 262: 4429-4432, 1987). Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant vector. Transfer of the construct may beperformed by any of the methods mentioned above which physically orchemically permeabilize the cell membrane. For example, Dubensky et al.(Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984) injected polyomavirusDNA in the form of CaPO₄ precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.

Anti-Sense

The term “antisense” is intended to refer to polynucleotide moleculescomplementary to a portion of a targeted gene or mRNA species.“Complementary” polynucleotides are those that are capable ofbase-pairing according to the standard Watson-Crick complementarityrules. That is, the larger purines will base pair with the smallerpyrimidines to form combinations of guanine paired with cytosine (G:C)and adenine paired with either thymine (A:T) in the case of DNA, oradenine paired with uracil (A:U) in the case of RNA. Inclusion of lesscommon bases such as inosine, 5-methylcytosine, 6-methyladenine,hypoxanthine and others in hybridizing sequences does not interfere withpairing.

Antisense polynucleotides, when introduced into a target cell,specifically bind to their target polynucleotide and interfere withtranscription, RNA processing, transport, translation and/or stability.Antisense RNA constructs, or DNA encoding such antisense RNA's, may beemployed to inhibit gene transcription or translation or both within ahost cell, either in vitro or in vivo, such as within a host animal,including a human subject.

The intracellular concentration of monovalent cation is approximately160 mM (10 mM Na⁺; 150 mM Kb). The intracellular concentration ofdivalent cation is approximately 20 mM (18 mM Mg⁺; 2 mM Ca⁺⁺). Theintracellular protein concentration, which would serve to decrease thevolume of hybridization and, therefore, increase the effectiveconcentration of nucleic acid species, is 150 mg/ml. Constructs can betested in vitro under conditions that mimic these in vivo conditions.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that effective antisense constructs may includeregions complementary to the mRNA start site. One can readily test suchconstructs simply by testing the constructs in vitro to determinewhether levels of the target protein are affected. Similarly,detrimental non-specific inhibition of protein synthesis also can bemeasured by determining target cell viability in vitro.

As used herein, the terms “complementary” or “antisense” meanpolynucleotides that are substantially complementary to the targetsequence over their entire length and have very few base mismatches. Forexample, sequences of fifteen bases in length may be termedcomplementary when they have a complementary nucleotide at thirteen orfourteen nucleotides out of fifteen. Sequences that are “completelycomplementary” will be sequences which are entirely complementarythroughout their entire length and have no base mismatches.

Other sequences with lower degrees of homology also are contemplated.For example, an antisense construct that has limited regions of highhomology, but also contains a non-homologous region (e.g., a ribozyme)could be designed. These molecules, though having less than 50%homology, would bind to target sequences under appropriate conditions.

Although the antisense sequences may be full length cDNA copies, orlarge fragments thereof, they also may be shorter fragments, or“oligonucleotides,” defined herein as polynucleotides of 50 or lessbases. Although shorter oligomers (8-20) are easier to make and increasein vivo accessibility, numerous other factors are involved indetermining the specificity of base-pairing. For example, both bindingaffinity and sequence specificity of an oligonucleotide to itscomplementary target increase with increasing length. It is contemplatedthat oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50 or 100 base pairs will be used. While all orpart of the gene sequence may be employed in the context of antisenseconstruction, statistically, any sequence of 14 bases long should occuronly once in the human genome and, therefore, suffice to specify aunique target sequence.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., Science, 260:1510-1513, 1993).

Alternatively, the antisense oligo- and polynucleotides according to thepresent invention may be provided as RNA via transcription fromexpression constructs that carry nucleic acids encoding the oligo- orpolynucleotides. Throughout this application, the term “expressionconstruct” is meant to include any type of genetic construct containinga nucleic acid encoding a product in which part or all of the nucleicacid sequence is capable of being transcribed. Typical expressionvectors include bacterial plasmids or phage, such as any of the pUC orBluescript™ plasmid series or viral vectors adapted for use ineukaryotic cells.

In preferred embodiments, the nucleic acid encodes an antisense oligo-or polynucleotide under transcriptional control of a promoter. The termpromoter will be used here to refer to a group of transcriptionalcontrol modules that are clustered around the initiation site for RNApolymerase II. Promoters are composed of discrete functional modules,each consisting of approximately 7-20 bp of DNA, and containing one ormore recognition sites for transcriptional activator or repressorproteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. A variety of specific eukaryotic promoter elements are known inthe art and any such known element may be used in the practice of theclaimed invention. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription. The particular promoter that is employed to control theexpression of a nucleic acid encoding the inhibitory peptide is notbelieved to be important, so long as it is capable of expressing thepeptide in the targeted cell.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. Any promoter/enhancer combination known in theart (e.g., the Eukaryotic Promoter Data Base) also could be used todrive expression of the gene.

Where a cDNA insert is employed, typically one will typically include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed, such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression construct is a terminator. These elements can serve toenhance message levels and to minimize read through from the constructinto other sequences.

In certain embodiments of the invention, the delivery of a nucleic acidin a cell may be identified in vitro or in vivo by including a marker inthe expression construct. The marker would result in an identifiablechange to the transfected cell permitting identification of expression.Enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic)or chloramphenicol acetyltransferase (CAT) (prokaryotic) may beemployed.

siRNA

Small interfering RNAs (siRNAs) are short RNA molecules (typically from21 to 23 nucleotides in length) that may be used to induce targeted genesilencing by RNA interference (Myers et al., Nature Biotechnology21:324-328, 2003; Elbashir, Nature 411:494-498, 2001; Caplen et al.,Proc. Natl. Acad. Sci. USA 98:9742-47, 2001). SiRNAs occur naturally invivo when double-stranded RNA is cleaved by ribonuclease III to producea short siRNA sequence. Synthetic siRNAs may also be introduced intocells to inhibit expression of one or more selected genes. SiRNAs may begenerated by standard solid-phase oligonucleotide synthesis, byRNA-specific endonuclease cleavage of double-stranded RNA, or byexpression of transfected DNA templates incorporating promoter sequencesfor RNA polymerase III. Introduction of siRNA into a mammalian cellresults in the targeted destruction of messenger RNAs of the samesequence. Commercial products for siRNAs are available from a number ofsources, such as Gene Therapy Systems, Inc. (San Diego, Calif.), Promega(Madison, Wis.) and Sirna Therapeutics (Boulder, Colo.).

Methods for design of siRNA sequences are publicly available. Forexample, the siRNA Target Finder may be used online at the Ambionwebsite. Target mRNA sequences are input into the program, which thenscans for 21 nucleotide sequences that begin with an AA dinucleotide.The program selects for siRNAs with about a 30 to 50% GC content,avoiding sequences with 4-6 polyT stretches that would function asterminators for RNA Polymerase III transcription. After selection of twoto four siRNA candidates, the generated sequences may be searched forhomology (for example, using the BLAST search engine on the NCBI server)to other untargeted mRNA sequences. SiRNAs with homology to non-targetedsequences are eliminated from consideration. SiRNA expression cassettesmay also be obtained from Ambion (Austin, Tex.). SiRNAs may be purchasedand used according to the manufacturer's instructions to providetargeted inhibition of the expression of specific genes, such as PDE-4and/or PDE-5.

Ribozymes

Another method for inhibiting the expression of specific genes withinthe scope of the present invention is via ribozymes. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forsterand Symons, 1987). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurekand Shub, 1992). This specificity has been attributed to the requirementthat the substrate bind via specific base-pairing interactions to theinternal guide sequence (“IGS”) of the ribozyme prior to chemicalreaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.,1990; Sioud et al., 1992). It was reported that ribozymes elicitedgenetic changes in some cells lines to which they were applied. Thealtered genes included the oncogenes H-ras, c-fos and genes of HIV. Mostof this work involved the modification of a target mRNA, based on aspecific mutant codon that is cleaved by a specific ribozyme.

Several different ribozyme motifs have been described with RNA cleavageactivity (Symons, 1992). Examples that are expected to functionequivalently include sequences from the Group I self splicing intronsincluding Tobacco Ringspot Virus (Prody et al., 1986), Avocado SunblotchViroid (Palukaitis et al., 1979; Symons, 1981), and Lucerne TransientStreak Virus (Forster and Symons, 1987). Sequences from these andrelated viruses are referred to as hammerhead ribozyme based on apredicted folded secondary structure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence that is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA—a uracil(U) followed by either an adenine, cytosine or uracil (A,C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

The large number of possible cleavage sites in genes of moderate size,coupled with the growing number of sequences with demonstrated catalyticRNA cleavage activity indicates that a large number of ribozymes thathave the potential to downregulate gene expression are available.Additionally, due to the sequence variation among different genes,ribozymes could be designed to specifically cleave individual genes orgene products. Designing and testing ribozymes for efficient cleavage ofa target RNA is a process well known to those skilled in the art.Examples of scientific methods for designing and testing ribozymes aredescribed by Chowrira et al., (1994) and Lieber and Strauss (1995). eachincorporated by reference. The identification of operative and preferredsequences for use in ribozymes targeted to specific genes is simply amatter of preparing and testing a given sequence, and is a routinelypracticed “screening” method known to those of skill in the art.

Formulations and Routes for Administration to Patients

In certain embodiments, the inhibitors or activators of PDE5, PKG, NOS,MMP or another protein and/or stimulators or agonists of cGMP may beused for therapeutic treatment of medical conditions, such as Peyronie'sdisease. Where clinical applications are contemplated, it will benecessary to prepare pharmaceutical compositions in a form appropriatefor the intended application. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

Aqueous compositions of the present invention comprise an effectiveamount of inhibitor or activator, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as innocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the inhibitors or activators of the present invention,its use in therapeutic compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions normallywould be administered as pharmaceutically acceptable compositions.

The active compounds also may be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts which are formed by reaction of basic groups withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed with free acidic groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

In this connection, sterile aqueous media which can be employed will beknown to those of skill in the art in light of the present disclosure.For example, one dosage could be dissolved in 1 ml of isotonic NaClsolution and either added to 1000 ml of hypodermoclysis fluid orinjected at the proposed site of infusion. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. The results disclosed below are generally addressed tothe following areas.

NO/cGMP

The antifibrotic effects of agents that, either orally or via genetransfer, stimulate the NO/cGMP pathways and increase NO levels and cGMPlevels or PKG activity or agents that inhibit oxidative stress bydecreasing ROS levels. For example, by a) gene transfer of the sensecDNA for iNOS or nNOS in a single transfection; b) long-termadministration of an oral NO donor (e.g. molsidomine), or the NOSsubstrate (L-arginine) that produces a continuously elevated level ofNO. Alternatively, by increasing cGMP and/or stimulating PKG by a)identifying PDE isoforms present in the affected tissue and using oralPDE inhibitors such as sildenafil, zaprinast, and pentoxifylline; b) bygene transfer of PKG1 cDNA, or its mutated version, to increase thelevel of PKG activation. In other alternatives, by reducing theconcentration of ROS by a) early (to arrest the development of thePD-like plaque, arteriosclerotic plaque or other fibrotic lesion) orlate (to induce regression of an already formed plaque) administrationof oral antioxidants such as vitamin E or S-adenosyl methionine (SAME);b) combination therapy with antioxidant (Vitamin E or SAME) combinedwith one or more NO donors (molsidomine or L-arginine) or cGMP/PKGtherapy (sildenafil, zaprinast, pentoxifylline, or PKG1 cDNA), to induceregression of the plaque (late treatment).

MMP

Stimulation of MMP (collagenolysis) induced by thymosin peptides orother MMP activators. Correlating MMP inhibition in fibrosis with thelevels of TIMP1, an inhibitor of MMP. Use of the MMP inducers thymosinβ-4 and 10 are to stimulate MMP activity.

Materials and Methods

Human Tissues and Cell Cultures

Human Tissue:

Human TA was obtained from non-PD patients (n=4), two undergoing partialpenectomy due to penile cancer and two undergoing penile prosthesissurgery. Plaque tissue was isolated from PD patients (n=8) who underwenta surgical procedure to treat this condition (Vernet et al., 2002;Ferrini et al., 2002; Magee et al., 2002b; Davila et al., 2003b).Fragments of newly obtained tissue are stored for 24 h in “RNA-later”(Ambion, Inc., Austin, Tex.), for RNA analysis, in 4% formalin forhistochemistry and immunohistochemistry, or in culture medium (DMEM/10%fetal calf serum) or fibroblast growth medium (FGM-2) (Clonetics,Walkersville, Md.) with 20% fetal bovine serum, for protein analysis orcell culture. Tissues were then frozen at −80° C. until further use,except for fixed portions that were stored at 4° C. in PBS untilparaffin embedding or cryosectioning, and pieces used for cell cultures.

Primary Human Cell Cultures:

Human fibroblast primary cultures were obtained from fragments of PDplaque or TA essentially according to Smith and Liu (2002), and theirpurity was established by immunohistochemistry, as detailed below. Newprimary cultures were obtained from fragments of PD plaque or TA thatwere washed in Hanks solution, minced in a fibroblast growth medium(FGM) (BioWhittaker Inc., Walkersville, Md.) and 20% fetal bovine serum(FBS), and plated onto a 25 cm² culture flask per specimen (Vernet etal., 2002). Fragments were left undisturbed until attachment for about 1week. Once the monolayer started to develop, the fragment was removed.Medium with 10% FBS was changed once a week and when cells achievedapproximately 80% confluence (3-4 weeks) they were trypsinized and splitonto 10 cm plates. Cells were allowed to grow again to 80% confluence,with medium changed twice a week. The cells collected from this passagewere considered as passage 1. Successive passages were performed at 1/3split ratio, and studies were carried out on cells from passages 3onwards. Studies were performed with PD cells from the 4^(th) to the10^(th) passages. Cells were incubated on: a) 75 cm² flasks for RNAisolation; b) 6-well plates for protein isolation; and c) 8-wellremovable chambers for cytochemistry and immunocytochemistry. Treatmentswith different additions were initiated 24 hours after plating andcontinued for different periods.

Cells incubated in 8-well chamber slides were allowed to grow to 50-60%confluence. At this point, cells received in duplicate sildenafil,pentoxifylline, or 8-Br cGMP at the concentrations indicated, and wereallowed to propagate for 3 days without changing medium. In certaincases SNAP was added and replaced daily after changing the medium(Vernet et al., 2002). All studies were done in duplicate or triplicate.For the isolation of rat TA fibroblasts, the TA was carefully dissectedfrom rat corpora cavernosa tissue, and cultures were developed and theirpurity tested as in the case of the human tissues.

Rodent Models and Tissue Processing

TGF-β1 Rat Model.

Male Fisher 344 rats, 9-11 month old purchased from the NIH/NIA colony(Harlan Sprague-Dawley, Inc., San Diego, Calif.) and maintained undercontrolled temperature and lighting, were anesthetized and injected inthe penile TA at the middle of the penis with either vehicle only(saline, group 1) or 0.5 μg recombinant human TGF-β1 (BiotechDiagnostic, Laguna Niguel, Calif., groups 2-5) as disclosed (Ferrini etal., 2002; Vernet et al., 2002). After the injection, groups 1 and 2were given drinking water while the other groups received water withL-arginine (2.25 g/kg/day, group 3) (Moody et al., 1997), or sildenafil(10 mg/kg/day, group 4) or pentoxifylline (10 mg/kg/day, group 5).Forty-five days later, or as indicated, animals were sacrificed andperfused through the left ventricle with saline followed by 4% formalin((Ferrini et al., 2002; Vernet et al., 2002). After the penises wereexcised, the penile skin was denuded by removing the glans and adheringnon-crural tissue. The penile shaft was separated from the crura and 2-3mm transverse slices were cut around the site of the saline or TGF-β1injection. All tissues were post-fixed overnight in 4% formalin, washedin PBS and stored at 4° C.

TGF-β1-iNOS Knock-Out Mouse Model.

The iNOS knockout strain (B6; 129PNOS2<Tm1Leu>), where iNOS expressionwas genetically blocked, and the corresponding wild type control (B6;129PF1/y) (Hochberg et al., 2000), were injected (2-3 months old) in theTA with TGF-β1 as in the rat and sacrificed 45 days later.

TGF-β1-Collagen I Promoter Mouse Model.

The transgenic line pGB 19.5/13.5 was obtained from George Bou-Gharios(London, England). These animals harbor the promoter of the α2 chain ofthe mouse collagen type I gene linked to the E. coli β-galactosidase,that is expressed in cells and tissues where collagen I is normallyexpressed (Fakhouri et al., 2001; Tharaux et al., 2000; Dussaule et al.,2000). Animals were injected into the TA as above with TGF-β1, andsacrificed.

Arterial Tree Rodent Model.

Young (3-month) and aged (22-24 month) male Brown Norway rats wereobtained from the NIH/NIA colony (Harlan Sprague-Dawley, Inc., SanDiego, Calif.), and maintained under controlled temperature andlighting. One half of the aged animals were treated for 3 weeks withL-NIL at 0.1 g/l in the drinking water, while the rest of the animalsreceived plain drinking water. Animals were anesthetized, pretreatedwith heparin, and perfused through the left ventricle with salinefollowed by 4% formalin. The abdominal aorta, brachial and femoralneurovascular bundles as well as the penis, denuded of its skin, wereremoved and post-fixed overnight in 4% formalin, and washed and storedin PBS at 4 C until paraffin embedding.

General Procedures

Injection-Electroporation.

Injection into the TA was performed with the appropriate AdV or plasmidcDNA constructs at doses described below, and electroporation wasapplied at 100 volts, 8 pulses/sec. 40 ms (Magee et al., 2002a).

Minipump Implantation.

Alza osmotic minipumps (Alza Corp, Palo Alto, Calif.), #2001D,delivering 8 ul/hr of a saline solution (100 ul) containing the selectedcompound during a period of 24 hs for “short-term” treatment, or 0.25ul/hour, for 2 weeks (Alza#1002) for “long-term” treatment, wereimplanted in a subcutaneous tunnel over the inguinal canal, and attachedto the abdominal muscles with a non-absorbable suture. A deliverycatheter from the minipump was placed through the tissues to the penilecrura and sutured to the perineal muscles, as previously described(Garban et al., 1997; Gelman et al., 1998).

Detection of PDE mRNA Expression in Tissues and Cells

Total RNA was isolated from the human TA and PD tissues, from theirrespective fibroblast cultures, and from rat TA and penile shafttissues, and their respective fibroblast and smooth muscle cellcultures, by the Trizol procedure (Gibco BRL, Gaithesburg, Md.). RNA wasthen submitted (1 ug) to reverse transcription (Vernet et al., 2002;Magee et al., 2002b; Ferrini et al., 2001b) using Superscript II RNaseH⁻ reverse transcriptase (Gibco BRL) and random primers (0.25 ug),followed by PCR with the respective gene specific primers (Kuthe et al.,2001): a) for human PDE5A, on nt 1027-1049 (forward) and nt 1788-1764(reverse) of the respective cDNA (Genbank #158526); encompassing a 762bp band common to the three variants 1-3; b) for rat PDE5A, the primerson nt 1905-1924 (forward) and nt 2479-2460 (reverse) of the respectivecDNA (Genbank #NM 133584), generating a 575 bp band; c, d) for humanPDE4A and B, on nt 942-965 (forward) and 1824-1802 (reverse), and nt1909-1931 (forward) and 2315-2292 (reverse), respectively, of the cDNAs(Genbank #NM 006202 and NM 002600, respectively); as the source of theexpected 883 bp (A) and 406 bp (B) bands; e) for rat PDE4, the primerson nt 241-260 (forward) and nt 656-637 (reverse) of the respective cDNA(Genbank #M25350), generating a band of 416 bp. PCR products wereseparated by electrophoresis on 1% agarose gels and stained withethidium bromide. For densitometry, normalization was performed againstthe GAPDH housekeeping gene fragment generated in the same PCR reaction.

Detection of PDE5 and 4 Protein Expression in Tissue and Cell Extracts

Tissue extracts were obtained by homogenizing in a 1:6 wt/vol ratio in abuffer containing 0.32 M sucrose, 20 mM HEPES (pH 7.2), 0.5 mM EDTA, 1mM dithithreitol and protease inhibitors (3 μM leupeptin, 1 μM pepstatinA, 1 mM phenylmethyl sulfonyl fluoride). In the case of cell extracts0.5 ml of this solution per 10 cm Petri dish was used. The particulateand cytosolic fractions were obtained by homogenization of the cells ina Polytron Homogenizer, (Brinkmann, Switzerland), and centrifugation at12,000×g for 60 min.

Equal amounts of protein (30 ug) were run on 7.5% polyacrylamide gels,and submitted to western blot immunodetection with polyclonal anti-mousePDE5 (against cGMP binding region) IgG (1:1000) (Calbiochem, La Jolla,Calif.), and a secondary donkey anti-mouse IgG linked to horseradish-peroxidase (Amersham Pharmacia, Piscataway, N.J.), followed by aluminol reaction (Simko and Simko, 2000; Magee et al., 2002b; Ferrini etal., 2001b). Human PDE5 does not cross-react with other PDE5 isoforms.Negative controls were performed without primary antibody.

For PDE4 immunodetection, the following affinity purified IgGs were used(FabGennix Inc., Shreveport, La.): a) anti-PDE4A selective antibody(detecting variants identified by 1, 5, 8, ×, and unassigned); b)anti-PDE4B (detecting variants 1-4), and anti-PDE4D (detecting variants1-5) (Salanova et al., 1999).

The presence of PDEs in the PD fibroblasts in culture was confirmed bythe ability of increasing concentrations of sildenafil andpentoxifylline to raise the basal cGMP and cAMP levels in triplicatewells, either in the absence or the presence of the NO donor, SNAP(S-nitroso-N-acetyl penicillamine) (Alexis Biochemicals, San Diego,Calif.) added daily at 100 lAM, as measured by cGMP and cAMP EIA (enzymeimmuno absorption) kits (Cayman Chemical, Ann Arbor, Mich.). Experimentswere performed in duplicate. Values were expressed as pmoles cGMP orcAMP/mg protein. To normalize for differences between experiments, thechanges in cGMP and cAMP levels exerted by sildenafil and pentoxifyllinewere expressed as % of their respective control values in the absence ofthe PDE inhibitors.

Histochemical and Immunohistochemical Determinations

In the case of cell cultures, at completion of incubations, slides wereremoved from the chambers and cells were fixed for immunodetection for20 min in 4% buffered formalin at room temperature for α-smooth muscleactin (ASMA) (as a myofibroblast marker), vimentin (as a generalfibroblast marker), and in certain cases for PDE5 and PDE4, or inethanol at −20 C for collagen I and III (Vernet et al., 2002). The cellswere quenched, blocked with normal goat serum and incubated withmonoclonal primary antibodies for ASMA and vimentin (SigmaImmunohistology Kits, Sigma Chemical Co, St. Louis, Mo.), collagen I,and collagen III (1:40) (Chemicon International, Temecula, Calif.),overnight at 4° C. (Vernet et al., 2002; Ferrini et al., 2002).Processing was according with the manufacturer's instructions for ASMA,vimentin and collagen, consisting in the respective monoclonalantibodies and an anti-mouse biotinylated secondary antibody, followedby avidin-biotinylated HRP and the 3-amino-9-ethylcarbazol (AEC)chromogen. For PDE5A, the antibodies were as described above. Negativecontrols omitted the first antibodies or were replaced by IgG isotype atthe same concentration of the first antibodies. Counterstaining was donewith Mayer's hematoxylin. All the slides were mounted with Aqua Mount(Lerner, Pittsburgh, Pa.). For PDE4, the anti PDE4A and PDE4B affinitypurified IgGs used for western blot was employed, and in addition theanti-PDE4A4 and anti-PDE4D (detecting variants 1-5) from the same source(FabGennix Inc.) were used.

In the case of tissue sections, the determinations of thecollagen/smooth muscle ratio were carried out with Masson trichrome(Ferrini et al., 2002; Davila et al., 2003b) on adjacent 5 μmparaffin-embedded cross-sections from the human normal tunical or plaquetissues, or from a 2 mm area around the site of injection in the ratsaline- and TGF-β1-injected shaft tissues. Other distal sections wereobtained along the rat penile shaft.

SMC and collagen fibers within the corporal tissue and vascular treewere estimated by Masson trichromic staining (Sigma Diagnostic, St.Louis, Mo.) (Ferrini et al., 2002; Vernet et al., 2002; Davila et al,2203b) in sections adjacent to those used for immunohistochemicalstaining, followed by image analysis to measure the ratio between SMC(red) and collagen fibers (blue). The results were expressed as red/blueratios per area (see below). In the arterial tree, the intima/mediathickness (IMT), and the diameter of the lumen were also measured.

The determinations of iNOS, nitrotyrosine, heme-oxygenase I, PAI-1(Davila et al., 2003b), manganese superoxide dismutase (MnSOD), and CuZnSOD (Cu/Zn SOD) (Martin et al., 1994) were carried out on 5 μmparaffin-embedded adjacent tissue sections, that were quenched forendogenous peroxidase activity after deparaffinization and rehydration.Sections were blocked with normal goat serum, and incubated withpolyclonal IgG antibodies against mouse iNOS (1:500) (TransductionLaboratories, Lexington, Ken.), nitrotyrosine (1:100) (Upstate, LakePlacid, N.Y.), Mn SOD and Cu/Zn SOD (Oxygen, Portland, Oreg.) (1:800 and1:500 respectively), heme oxygenase I (Stressgen, San Diego, Calif.), orPAI-1 (Abcam Ltd, Cambridge, UK). For negative controls the firstantibodies were replaced by IgG isotype. The detection was based on asecondary anti-rabbit biotinylated antibody (1:200) for iNOS andnytrotyrosine (Calbiochem, La Jolla, Calif.), or anti-sheep biotinylatedantibody (1:200) for Cu/Zn and Mn SOD, followed by the ABC complex(1:100) (Calbiochem) and 3,3′ diaminobenzidine (spelling) (DAB) (Sigma,St Louis Mo.). Sections were counterstained with hematoxylin.

TUNEL Assay for Apoptosis

The TUNEL assay (Ferrini et al., 2001a, 2001b) was performed in theadjacent matched tissue sections used for collagen, iNOS ornitrotyrosine staining, applying the Apoptag Oncor kit (Oncor,Gaithersburg, Md.). In brief, after deparaffinization and rehydration,sections were incubated with proteinase K (20 ug/ml) and endogenousperoxidase activity was quenched with 2% H₂O₂. Sections were incubatedwith digoxigenin-conjugated nucleotides and TdT, and subsequentlytreated with antidigoxigenin-peroxidase. To detect immunoreactive cells,sections were stained with 0.5% DAB/0.01% H₂O₂, and counterstained with0.5% methyl green. As a negative control, buffer was substituted for theTdT enzyme. Testicular sections from old animals were used as positivecontrol. For cell cultures, the cells were fixed in 4% formaldehyde for30 min on ice, and post-fixed with ethanol-acetic acid 2/1 for 5 min at−20 C. Then the above procedure was applied, except that the proteinaseK was omitted.

Quantitative Image Analysis (QIA)

The quantitation of the staining obtained by either histochemical orimmunohisto/cytochemical techniques was performed by computerizeddensitometry using the ImagePro 4.01 program (Media Cybernetics, SilverSpring, Md.), coupled to an Olympus BHS microscope equipped with a SpotRT digital camera or VCC video camera (Wang et al., 2001; Ferrini etal., 2001a, 2001b; Davila et al., 2003b).

The number of positive cells was counted in a computerized grid againstthe total number of cells determined by counterstaining, and resultswere expressed as a percentage of positive cells over total cells. Inaddition, the integrated optical density (IOD) was obtained by measuringthe density per object and multiplying it by the respective area. Thesum of all the individual values in the field was then divided by thenumber of positive cells, to obtain the mean IOD/positive cell, as ameasure of average immunoreactivity/cell. In certain cases, results wereexpressed as the unweighted average optical density per area (O.D/AREA),to determine the relative concentration of immunoreactive antigen. Forcollagen/smooth muscle staining, the ratio between the width of the areastained positive for collagen (blue) divided by the total area of thelacunar spaces plus smooth muscle (white+red) was employed. Theapoptotic index (rate of programmed cell death) was calculated as thepercentage of apoptotic cells within the total number of cells in agiven area (non-apoptotic nuclei plus apoptotic cells). In all cases,five non-overlapping fields were screened per tissue section or perwell. Three sections per tissue specimen from groups of five animals, ortwo wells per experimental point in cell incubations, were then used tocalculate the means+/−SEM.

For iNOS, nitroyrosine, heme-oxygenase, PAI-1, MnSOD and Cu/Zn SODdetermination, at least 6 sections per specimen were analyzed. Eachslide assayed had its corresponding negative control. In certain cases,the number of immuno-positive cells was determined as a percentage ofthe total counterstained nuclei in a computerized grid. In the Massonstaining, the ratio between SMC (red) and collagen fibers (blue) wasobtained and expressed per area. The rate of programmed cell death(apoptotic index) was expressed as the percentage of apoptotic cellswithin the total number of cells in a given area (non-apoptotic nucleiplus apoptotic cells).

Statistical Analysis

Values were expressed as mean (M)+/−standard error of the mean (SEM).The normality distribution of the data was established using theWilk-Shapiro test, and the outcome measures between two groups werecompared by the t test. Multiple comparisons among the different groupswere analyzed by a single factor analysis of variance (ANOVA), followedby post-hoc comparisons with the Student-Neuman Keuls test, according tothe Graph Pad prism V. 30. Differences among groups were consideredsignificant at P<0.05.

Example 1. Spontaneous iNOS Induction In Vivo in the PD Plaque Leads toIncreased NO Synthesis, Peroxynitrite Formation, and FibroblastApoptosis

It has been reported that aging per se results in the spontaneousinduction of iNOS and the formation of the NO metabolite, peroxynitrite,in both the rat hypothalamus (Ferrini et al., 2001b; Vernet et al.,1998) and corpora cavernosa (Ferrini et al., 2001a). This wasaccompanied by apoptosis of both the neurons and the cavernosal smoothmuscle. In the TGF-β1 induced rat model of PD, a similar iNOS inductionin the TA (Bivalacqua et al., 2000; Hellstrom, 2001) has been reported.Initially, it was assumed that this process of iNOS induction wasdeleterious to the TA.

As proposed herein, it is believed that iNOS induction in the TA, andperhaps in fibrosis in general, is a beneficial, anti-fibrotic, cellulardefense mechanism. The locally produced NO from elevated iNOS wouldinhibit collagen deposition, oppose pro-fibrotic agents, and induceapoptosis of myofibroblasts, which pathologically persist in the PDplaque. This model has been examined in the TA (Ferrini et al., 2002;Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al.,2002) by quantitative image analysis (QIA) of immunohistochemical andhistochemical stained tissue sections, and measurement of RNA andprotein expression by quantitative RT/PCR, northern blots, westernblots, DNA microarrays, and other procedures (n=5 to 9 per experimentalgroup). All results discussed below are significant (p<0.05), unlessstated otherwise.

It was first observed that in the human PD plaque, iNOS induction asseen by immunohistochemistry occurs spontaneously in discrete cells(Ferrini et al., 2002). These cells were identified as fibroblasts andmyofibroblasts based on vimentin as markers for both cell types andalpha smooth muscle actin (ASMA) for myofibroblasts (Vernet et al.,2002). iNOS expression as measured by immunohistochemistry was alsodetected in the rat PD-like plaque 45 days after TGF-βI injection in theTA, in comparison to control tissue obtained from rats injected withsaline (Ferrini et al., 2002; Vernet et al., 2002). In addition, inplaque tissue from both rat and human, iNOS induction was accompanied byincreased peroxynitrite, a product formed by the reaction of NO with ROS(Ferrini et al., 2002). In contrast to its inductive effects on cellapoptosis, peroxynitrite does not induce collagen deposition orfibrosis, which means it is not pro-fibrotic per se (Okamoto et al.,1997), and therefore, differs considerably from one of the compoundsfrom which it originates, ROS, that is highly pro-fibrotic (Casini etal., 1997; Muriel et al., 1998a; Hung et al., 1995; Poli, 2000; Curtinet al., 2002; Cattell, 2002; Kim et al., 2001).

The fibrotic plaque was visualized in the rat model by Masson stainingshowing disorganization of collagen fibers and intensification ofcollagen deposition and thickening of the TA (Ferrini et al., 2002). Inthe case of the human plaque, similar changes were revealed with Massonstaining. We further observed an increase in collagen I mRNA levels andprotein-bound hydroxy-proline, which are additional direct measurementsof tissue collagen content (Ferrini et al., 2002). These initial resultsdemonstrated that iNOS was strongly expressed in PD tissue and may playan important role in the plaque.

To examine the specific role of NO on PD plaque formation, overall NOSactivity was increased in the TA by treating the animals with the oralNOS substrate, L-arginine. The long-term oral administration of the NOSsubstrate, L-arginine, in the drinking water (2.25%), leads to astimulation of NOS activity and NO synthesis in the whole penis (Moodyet al., 1997) and should also be increased in the TA. In addition, NOhas been shown to be a down-regulator of collagen synthesis (Haig etal., 1994). If NO has a down-regulatory effect on collagen synthesis inthe plaque, then overproduction of NO should inhibit this collagensynthesis and prevent development or halt progression of the plaque. Itwas observed that increasing NO by oral L-arginine treatment for 45 daysled to a considerable decrease in the size of the TGF-β1-induced plaque(FIG. 2A, top panels). The plaque in the rat model consists ofdisorganized collagen fibers and thickening of the TA that seems tospread extensively from the site of the TGF-β1 injection. This observeddecrease in plaque development by oral L-arginine treatment was alsoverified by quantitating the ratio of the areas in the TA occupied bycollagen fibers to the areas occupied by the cells and lacunar spaces(FIG. 2B). Changes in the width of the tunica as measured by QIA alsoconfirmed these results (not shown). These observations demonstrate thatNO, from oral NO donors, seems to play an anti-fibrotic role in the TAof the TGF-β1 injected rat model of PD.

Another major effect of high levels of NO in any tissue is itsconversion, by its interaction with ROS, into peroxynitrite, which is aknown inducer of apoptosis (Beckmann et al., 1996; Ferrini et al.,2001a; Vernet et al., 1998; Heigold et al., 2002; Duffield et al., 2000;Zhang et al., 1999). NO in the TA may also act to increase apoptosis ofthose cells within the PD plaque that are responsible for promotingcollagen synthesis. In the PD animal model given oral L-arginine(2.25%), there appeared to be an increase in the number of apoptoticcells per field in the PD-like plaque as compared to the control animals(FIG. 3A, top panels). But when an apoptotic index (apoptoticcells/total number of cells) was used to quantify apoptosis, nosignificant difference was observed (FIG. 3B), because of the parallelincrease in cell number.

Example 2. Inhibition of iNOS Activity In Vivo Stimulates Both CollagenSynthesis and Collagen Fiber Deposition in the Rat PD-Like Plaque

The studies with L-arginine detailed in Example 1 show modulation of thesize of the PD plaque by NO. To determine whether the NO involved inthis anti-fibrotic process emanated from iNOS, we studied in the TGF-β1rat model the effects of specifically blocking iNOS activity by thelong-term oral administration of L-NIL, a specific iNOS inhibitor(Ferrini et al., 2002, Vernet et al., 2002). In the TGF-β1 injected ratmodel, treatment with L-NIL, which lowers NO derived from iNOS, induceda remarkable expansion and thickening of the TA that was due toexcessive collagen fiber deposition (Ferrini et al., 2002). We alsoobserved a considerable increase in peroxynitrite as indicated bynitrotyrosine formation in the TA (Ferrini et al., 2002). Theseobservations further support the role of NO from iNOS in reducing thegrowth of the plaque in the rat TA.

The effect of L-NIL in increasing collagen in the TA of the TGF-β1 ratmodel may be due to an increase in collagen synthesis, a decrease in itsnormal breakdown, or both. To determine whether the larger PD plaque inthe L-NIL treated rat is due, at least in part, to an increase incollagen I synthesis (the most prevalent collagen protein in the TA),and not simply to the inhibition of collagenolysis by the MMPs, weinjected a cDNA plasmid construct of the collagen I promoter driving theexpression of a reporter gene (Magee et al., 2002a) into the site of theoriginal TGF-β1 or saline injection, 10 days prior to sacrifice and 35days after the TGF-β1 injection. This plasmid is an indicator ofcollagen I transcriptional activity within the rat PD plaque. Expressionof the reporter β-galactosidase, measured by luminometry in tissueextracts from areas at and around the plaque, was considerablyintensified in comparison to the control TA (Vernet et al., 2002). Thissuggests that the reduction in NO by L-NIL inhibition of iNOS, directlyor indirectly, activates pro-fibrotic factors such as ROS to furtheractivate the collagen I promoter.

Example 3. The Inhibition of PDE Activity In Vivo Reduces CollagenDeposition and Intensifies Fibroblast Apoptosis in the PD-Like Plaque inthe Animal Model

Numerous studies have documented that increasing the levels of cGMP byinhibition of PDE enzymes, either with non-specific PDE inhibitors, suchas pentoxifylline (Corbin and Francis, 1999; Uckert et al., 2001;Fischer et al., 2001; Desmouliere et al., 1999; Kremer et al., 1999), orspecific isoform inhibitors for PDE-5 such as exisulind (Chan et al.,2002; Takuma et al., 2001), can inhibit collagen synthesis and fibrosisand can induce apoptosis in vivo and in cultured cells (Chiche et al.,1998; Pandey et al., 2000; Tao et al., 1999; Loweth et al., 1997;Sirotkin et al., 2000; Taimor et al., 2000; Schade et al., 2002; Horioet al., 1999; Thompson et al., 2000). Thus, elevating cGMP levels may beable to inhibit tunical plaque formation. A study was performed todetermine whether the antifibrotic effects of NO in human and rat PD maybe at least partially mediated by the elevation of its downstreamproduct, cGMP.

Pentoxifylline (a non-specific, generalized PDE inhibitor), andsildenafil (specific PDE-5 inhibitor) were given orally to the rat intheir drinking water (100 mg/l for each PDE inhibitor) for 45 daysfollowing TGF-β1 injection to initiate the plaque in the rat model. FIG.2A, bottom panels shows that, as assessed by Masson staining in terms ofcollagen fiber/cell-lacunae area ratio, and width of the tunica (notshown), there was considerable reduction in plaque size induced by thePDE inhibitors. This was accompanied by an intensification of theapoptotic index, especially for pentoxifylline (FIG. 3A, bottom panels).The finding that pentoxifylline is more effective than sildenafil ininducing apoptosis may be due to a number of possible mechanisms. Sincepentoxifylline inhibits multiple PDE isoforms, it may suggest that otherPDEs besides PDE5 may play a role in elevating cGMP levels within the TAthat will lead to apoptosis of cells within the plaque. Additionally,PDE inhibitors may act differentially on the three processes thatultimately would inhibit fibrosis development, namely myofibroblastapoptosis, collagen synthesis, and collagen degradation. Although wehave not identified the cells undergoing apoptosis, it is likely thatthey are fibroblasts/myofibroblasts, because those are the predominantcellular component of the rat TA, and because of the directdemonstration of the effects of these treatments on cultures of humanfibroblasts and myofibroblasts (see below).

Example 4. Presence of PDE5 in the Human Penile Tunica Albuginea and PDPlaque, in the Rat Tunica Albuginea, and in Fibroblasts Cultured fromthese Tissues

As an initial assessment of PDE isoforms expressed in the TA, RT-PCR wasperformed on splice products of PDE5A (Kim et al., 2000). The observedeffects of sildenafil (specific PDE5 inhibitor) and to a partial extentpentoxifylline (has some PDE5 inhibitor activity) are probably mediatedby the PDE5A isoform. RT/PCR with primers common to the 3 PDE5A variants(Uckert et al., 2001; Lin et al., 2000a, 2002a) have shown that thisenzyme is expressed in both human TA and PD tissues, as well as controltissue, the corpora cavernosa (FIG. 4A). FIG. 4A shows the ethidiumbromide staining of PCR DNA fragments from reactions carried out induplicate and fractionated by agarose gel electrophoresis. The 575 bpPDE5A DNA band was generated as expected from the rat penile shaft (PS)and the 762 bp from the human corpora cavernosa (CC) RNAs, and wasamplified to a similar level in total RNA from the human TA and PD. NoRNA was extracted from the normal TA and the TGF-β1-induced PD-likeplaque in the rat, due to the difficulty in dissecting large amounts oftissue to avoid contamination by cavernosal smooth muscle.

PDE5A expression was confirmed at the protein level by western blotassays of tissue extracts, as shown by the luminol-stained protein bands(FIG. 4B), that can discriminate the three splicing variant proteins ofPDE5A designated as 1, 2, and 3 with respective apparent sizes of 100,92, and 83 kDa, respectively (Lin et al., 2000a, 2002a). The threevariants were detected as expected in the rat cerebellum (CER), ourcontrol tissue, whereas in the rat penile crura (CRU) and shaft (PS),the predominant forms were the 1 and 3, respectively, with only tracesof variant 2 in the crura, and a band smaller than the 3 variant in thepenile shaft. This PDE5A-3 variant, accompanied by smaller amounts ofthe 2 variant, was also expressed in the human corpora cavernosa (CC),and in the TA and PD plaque. Some PDE5A-1 variant was also detected inthe human CC.

Immunohistochemistry in human PD and TA sections confirmed theexpression of PDE5 in both tissues, at a higher level in PD. In the rat,it is extremely difficult to isolate pure TA and PD-like tissue totallyfree from corpora cavernosa cells. Therefore, the whole rat penileshaft, comprised of both TA and corpora cavernosa was assayed.Immunocytochemical detection with an antibody detecting all threevariants of PDE5A revealed that it was expressed in discrete cellsinterspersed among collagen fibers in the normal human TA and the PDplaque (FIG. 4C, upper panels). PDE5A was also detected in the media ofthe of the dorsal artery and those within the corpora cavernosa, and inboth the corporal smooth muscle and TA of the penis (FIG. 4C, lowerpanels)

PDE5 mRNA was also identified by RT/PCR in the fibroblasts cultured fromthe human normal TA and PD plaque, and from the rat TA (FIG. 7A), andthe respective protein was detected by western blot in the human cellsas a single PDE5A-3 variant, which agrees with what was observed in vivoin the TA and PD plaque, (FIG. 7B). The rat TA fibroblasts also expressthe 3 variant, accompanied by equal amounts of the 1 variant, despitethe latter larger variant was not detected in the rat penile shaft.Immunocytochemical detection (FIG. 4C, upper panels) confirmed theexpression of PDE5A in the three types of cells, namely fibroblasts fromthe human normal TA and PD plaque, and from the rat normal TA. However,in the latter case, as opposed to the human cell cultures derived fromtissues reasonably free from contamination by cavernosal smooth muscle,the rat fibroblasts were obtained from whole corpora cavernosa includingthe smooth muscle. By successive passages in fibroblast culture medium(Smith et al., 2002), rather pure fibroblast cultures were selected, asevidenced from vimentin staining, and some were myofibroblasts, as seenwith ASMA staining (FIG. 7A, bottom panels).

Example 5. Presence of PDE4 Variants in the Human Penile TunicaAlbuginea and PD Plaque, in the Rat Tunica Albuginea, and in FibroblastsCultured from these Tissues

PDE4 mRNA Studies

Since the cAMP-dependent PDE inhibitor, pentoxifylline, has been usedpreviously as an antifibrotic compound (Lee et al., 1997; Becker et al.,2001; Raetsch et al., 2002), we investigated by RT/PCR whether PDE4 isexpressed in the TA and PD tissues and the respective cell cultures,utilizing primers for two (A and B) of the three variants. FIG. 12Ashows that PDE4A and B mRNAs are expressed in the human normal TA and inthe PD tissue. Both variants were also detected in human corporacavernosa tissue containing mainly smooth muscle (not shown). Confirmingthese results, PDE4A and B mRNAs were also found in the fibroblastscultured from human TA and PD (FIG. 12B). In the case of the rat, PDE4mRNA (without variant discrimination) was detected in the TA cells, andto a lesser degree in the penile shaft tissue, thus suggesting that PDE4in the rat TA fibroblast cultures does not arise from contamination withsmooth muscle, which in any case had been reasonably excluded above byimmunocytochemistry.

PDE4 Western Blots

Confirmation of the expression of PDE4A at the protein level wasobtained by western blot with an antibody for the different variants, inextracts from the human cultured cells used for the identification ofPDE4A in TA and PD plaque. FIG. 12C shows an intense 76 kDa band thatwould correspond to a variant identified in testis (Salanova et al.,1999), as well as a minor 102 kDa band for the so-called PDE4Ax, alsoseen in the testis. The 76 kDa protein is very intense in the threehuman tissues: TA, PD plaque, and corpora cavernosa, but the 102 kDaband was virtually not detected. No PDE4B could be visualized when thewestern blot membranes were stripped and reacted with an antibodyspecific for this isoform (not shown).

PDE4 Immunohistochemistry

Immunodetection with the PDE4A antibody identified cells all along theinternal side of the TA, as well as in the corpora cavernosa smoothmuscle, expressing PDE4A (FIG. 13, top). An antibody specific for PDE4Dalso showed cells reactive for this isoform, evidencing that both PDE4genes are expressed in the TA and corpora cavernosa (FIG. 13, top). Asimilar situation is seen in the rat TA fibroblasts in culture, withconsiderable expression of PDE4A in most cells, whereas only a fractionof the cells express PDE4D (FIG. 13, middle). In contrast, most of thehuman TA fibroblasts were intensively stained with antibodies againstthe A and D isoforms (FIG. 13, bottom). Despite the fact that the PDE4BmRNA was identified by RT/PCR (see FIG. 12), virtually no proteinreactivity for this isoform was observed by immunodetection in tissuesections or cell cultures (not shown). A similar situation occurred withone of the variants of PDE4A (PDE4A4), utilizing a specific antibody,different from the general used above for PDE4A that according to thesupplier does not detect variant 4.

Example 6. Incubation of PD Fibroblast Cultures with PDE Inhibitors or acGMP Analog Reduces Collagen I Synthesis and MyofibroblastDifferentiation, and Increases Apoptosis

Verification that the PDE5A and PDE4 proteins detected in the TA and PDcells and tissues are enzymatically active was obtained by measuring thelevels of cGMP in cell extracts of the PD fibroblast cultures, with abasal mean value+/−SEM of 5.0+/−0.4 pmol/mg protein (n=5) in the absenceof additions. Levels of cGMP increased 5.0-fold with 100 uM SNAP (an NOdonor) for 3 days, with fresh daily replacement of medium with SNAP. AcGMP analog able to enter the cell, 8 Br-cGMP, at 10 μM, was also ableto increase cGMP levels by 6.4-fold, and with 100 μM 8 Br-cGMP, thelevels of cGMP were dramatically elevated by 38.7-fold. The basal levelsof cAMP were 42.6+/−12.7 pmol/mg protein in the absence of SNAP and didnot vary significantly when measured after 3 days with SNAP.

The cGMP-dependent PDE5 inhibitor sildenafil did not significantlystimulate cGMP levels in the absence of SNAP (not shown). However, inthe presence of the NO donor, the cGMP levels expressed as % of thebasal control levels in the absence of sildenafil, were increaseddose-dependently by sildenafil after the 3-day incubation, as expected(FIG. 14A). When cells were incubated with this NO donor, increasingconcentrations of pentoxifylline, also as expected, did not increasesignificantly cGMP levels expressed as % of control levels (FIG. 14B),but were very effective in increasing cAMP levels (FIG. 14C), thusconfirming its role as a cAMP-dependent PDE inhibitor with little or noeffect on cGMP-dependent PDE.

In order to determine whether the PDE inhibitors may reduce collagensynthesis, the PD cells were incubated with or without the drugs atlower concentrations: pentoxifylline at 200 nM, and sildenafil, at 50and 200 nM. After 3 days, cells were fixed and the intracellulardeposition of collagen I and III was determined by immunocytochemistrywith specific antibodies against the two isoforms. The antibody againstcollagen I elicited an intense granular and perinuclear staining (notshown). In contrast, collagen III was detected in only in about 30% ofthe cells, and stained more diffusely and rather lightly, even whencells where treated with TGF-βI (10 ng/ml), a known stimulator ofcollagen III synthesis (FIG. 5B).

Quantitation by image analysis (FIG. 5A) in the cultured human PDfibroblasts indicated that in the absence of additions, most of thecells (100%) expressed collagen I, and that both pentoxifylline andsildenafil at 200 nM completely inhibited collagen synthesis in a smallnumber of cells (5-15% of the total, FIG. 5A top), and significantlyreduced (30-40% decrease) the average intensity of expression per cell(FIG. 5A, bottom). In contrast, the PDE inhibitors did not decrease, buteven increased, the synthesis of collagen III (not shown). Moredrastically than in the case of collagen I, both of the PDE inhibitors(pentoxifylline and sildenafil) significantly reduced the number of ASMApositive cells (myofibroblasts) from 37% in the control to about 24%.The average ASMA expression per cell was significantly reduced by thePDE inhibitors by more than 90% in all cases.

To show that in the case of sildenafil some of the effects are mediatedby the elevation of cGMP, the PD fibroblasts were then incubated for 3days with 8-Br-cGMP, and a significant (30%) reduction in the number ofcells expressing collagen I was obtained at 10 μM 8 Br-cGMP (FIG. 6),although a higher concentration (400 μM) did not induce further decrease(FIG. 6). In contrast to the effects of the PDE inhibitors observed inthe previous experiments, collagen I expression per cell was reducedonly moderately and non-significantly by the cGMP analog. Thedifferentiation of fibroblasts into myofibroblasts measured by the levelof ASMA expression per cell was decreased significantly by 400 μM 8Br-cGMP, as in the case of the PDE inhibitors, but there was no effecton the relative number of positive cells (FIG. 6).

The increase of cGMP in the PD cells incubated with 8 Br-cGMP leads to astimulation of apoptosis, as shown by an increase in apoptotic bodiesdetected with the TUNEL technique (FIG. 21A). However, because ofvariability between experiments, the considerable 2.3 fold-increasemeasured by image analysis did not achieve statistical significance(FIG. 21B).

Example 7. Increase in NO Levels in Fibroblast Cultures from HumanNormal TA and PD Leads to Peroxynitrite Formation, Fibroblast Apoptosis,and Reduction of Intracellular Collagen

We have developed primary cell cultures from human normal TA and PDplaque, obtained from different patients, that were dissected to avoidcontamination with corporal smooth muscle. These cultures containfibroblasts and some myofibroblasts, as shown by a 100%immuno-reactivity with a vimentin antibody (Vernet et al., 2002) andrepresent the main cellular component of the original tissues.

The main features of these cultures, demonstrated by QIAimmunocytochemistry, are: 1) a substantial morphological differencebetween the TA and PD plaque cells (Vernet et al., 2002), thatcorresponds to the observations in vivo. PD cells change from small,more spindle shaped cells to much bigger, polygonal cells with biggernuclei and expansions, and in certain cases typical “stellate”appearance whereas TA cells do not substantially change in morphology.2) These fibroblasts, particularly those of PD origin, are able todifferentiate into vimentin+/ASMA+myofibroblasts comprising about 30% ofthe cells in culture (Vernet et al., 2002), and this percentage ofmyofibroblasts is also seen in vivo in the plaque (Vernet et al., 2002).3) The cultures can be induced to express iNOS, synthesize collagen I,and undergo apoptosis both in vitro and in vivo (Vemet et al., 2002). 4)Their responses to different agents, specifically the inhibitory actionof an NO donor, SNAP, on a) collagen I synthesis in all the cells, andb) myofibroblast differentiation (Vernet et al., 2002), and theirresponse to PDE inhibitors in cell culture (FIG. 5) resemble theresponses observed in vivo in the animal model of PD treated with NOdonors or PDE inhibitors (FIG. 3). 5) Collagen III, is also synthesizedbut to a lower extent than collagen I and is not significantly affectedby NO or PDE inhibitors (FIG. 5).

The use of an NO donor SNAP or iNOS induction with a cytokine cocktailincreased intracellular NO reduced fibroblast numbers in both normalhuman TA and PD cultures (Vernet et al., 2002). This process isassociated with the production of peroxynitrite, as evidenced bynitrotyrosine formation (not shown) and resembles the increase inapoptosis seen in vivo after sildenafil and pentoxifylline treatments.

Example 8. The Increase in cGMP Levels in Fibroblast Cultures Leads toFibroblast Apoptosis and the Reduction in Intracellular Collagen I

To verify that the effects of pentoxifylline and sildenafil describedabove were due to an elevation in cGMP levels, we incubated human PDcells with a stable cGMP analog able to traverse the cell membrane,8-BrcGMP. This compound (with levels as low as 10 uM) inhibited collagenI production by the cells (FIG. 6), and increased apoptosis, asdetermined by TUNEL (Ferrini et al., 2001a; Vernet et al., 1998), from7.5+/−0.4 (control) to 20.5+/−1.1 (100 uM 8-BrcGMP) cells per field. Theintracellular cGMP levels increased from 0.02 to 1.83 nmoles/10⁶ cellswith 100 uM 8-BrcGMP in control vs. treated cells, respectively.

The effects of the PDE inhibitors, as seen in in vivo experiments, aremost likely mediated in part by PDE-5, as shown by the presence of PDE5AmRNA (FIG. 7A), and specifically, the PDE5A-3 protein variant as in thein vivo derived tissues (FIG. 7B), in both the human TA and human PDderived cells. The fibroblast cultures obtained from the rat TA expressall three PDE-5 variants. All human and/or rat cell cultures of normalTA and PD tissues were PDE5 positive by immunocytochemistry (FIG. 7C,top panels). Cells derived from human TA tissue were clearly fibroblastswith some myofibroblast differentiation, as assayed with vimentin andASMA markers (FIG. 7C, bottom panels).

Example 9. ROS Levels are Increased in the Human and Rat PD-Like PlaqueTissues and are Reduced by NO

ROS plays an important role in the development and maintenance of manyfibrotic disorders including PD, by stimulating collagen synthesis(Poli, 2000; Curtin et al., 2002; Cattell, 2002; Kim et al., 2001; Fanet al., 2000; Higuchi et al., 1999). Therefore, the interplay andreactivity of ROS with NO may be an important therapeutic target. Wehave previously shown that heme-oxygenase I immunoreactivity, a markerfor the strong pro-fibrotic factor ROS, is increased in the PD plaque incomparison to normal TA in the human and the TGF-β1 rat model of PD(Ferrini et al., 2002). Additionally, when iNOS activity was blockedwith L-NIL in the TGF-β1 rat model, there was a considerable elevationof ROS levels (Ferrini et al., 2002). The same inverse correlationbetween NO and ROS was observed utilizing superoxide dismutaseimmunodetection, which measures the antioxidative response in both humanand PD tissue and in human fibroblast cultures (not shown). Thisreduction in the NO/ROS ratio is associated with a considerablestimulation of collagen deposition and collagen synthesis (Ferrini etal., 2002).

Example 10. Use of Gene Transfer and Reporter Gene Expression forAnalyzing PD

Several of the therapeutic approaches disclosed herein are based on genetransfer of cDNA constructs to the penile TA (e.g., iNOS, PKG).Therapeutic administration of recombinant cDNA may lead to an elevatedexpression of the corresponding anti-fibrotic protein. In the case ofthe penis, we have utilized plasmid and adenoviral constructs of iNOSand penile nNOS (PnNOS) (Magee et al., 2002a), including the use ofplasmid and adenoviral constructs expressing β-galactosidase as areporter gene, and electroporation to enhance viral and plasmid uptakeduring transfection (Magee et al., 2002a).

Such constructs can penetrate and spread into the TA, as shown by X-galstaining (FIG. 8) suggesting that the direct injection to the TA withand without electroporation is feasible for targeting genes to the TAfor arresting or reversing the growth and development of the PD plaque.

Example 11. Confirmation of the Pro-Fibrotic Role of TGF-β 1 Expressionin Another Model of PD

The TGF-β1 rat model for PD is a very valuable tool, and since itsintroduction in 1997 (El-Sakka et al., 1997b, 1998, 1999), we have beenable to study various aspects of the pathophysiology of PD, some ofwhich are presented above. However, in a different experimental designbased on the ubiquitous finding of fibrin in histological samples ofhuman PD tissue (not shown), we have recently re-confirmed theimportance of TGF-β1 as the main profibrotic factor in eliciting thePD-like plaque in the TA of the rat. We observed considerable expressionof this factor in a tunical lesion induced by the injection of apreparation of human fibrin (fibrinogen/thrombin/aprotinin) in the ratTA (FIG. 9). This tunical lesion, caused by fibrin and mediated byTGF-β1, is fully developed at 3 weeks, as visualized by Masson staining,and is indistinguishable from the one caused by the injection of TGF-β1alone. However, the TGF-β1 model requires 6 weeks to develop, whereasthis fibrin induced model only requires 3 weeks to fully develop (notshown). The fibrin induced plaque is accompanied by detection of fibrinin the lesion (similar to what is seen in the human, but absent from theTGF-β1 injected rat model), disorganization of elastin fibers,expression of iNOS and heme-oxygenase I, and an increased level ofapoptosis. Save for the presence of fibrin in the TA, all findings aresimilar to the ones observed in the TGF-β1 injected model (not shown).

Example 12. The TA and the PD Plaque are Tissues in Constant Turnover,and Collagenase Inhibition May Play a Role in Collagen Accumulation

As disclosed above, collagen synthesis is stimulated in the TGF-β1animal model of PD, particularly when NO synthesis is partiallyinhibited by L-NIL. Data obtained by DNA microarray analysis (Clontech)has allowed us to define and compare changes in the profile of multiplegene expressions at the mRNA level in the PD plaque versus normal TA.Data (Table 1), obtained in 9 patients and 9 control subjects indicatedthat: a) PD, like its related disorder Dupuytren's contracture, is acondition that seems to be in a state of dynamic flux, with alterationsin the expression of mRNAs for genes related to collagen turnover andtissue remodeling, extracellular matrix synthesis and degradation, andcell replication and apoptosis. This suggests that the fibrosis of PD isnot a terminal event but a dynamic one, and that it is possible topharmacologically affect its steady state and alter its direction by: a)inhibiting collagen synthesis and/or fibroblast differentiation andreplication, since a subset of differentially expressed genes arerelated to these processes; and/or b) by stimulating collagen breakdown,since there is a considerable increase in the expression of differenttypes of MMPs (e.g. MMP2 and MMP9). The increased expression of MMPs wasconfirmed in human PD by RT/PCR (FIG. 10).

MMPs may play an important role in extracellular matrix remodeling inthe PD plaque. The inhibition of MMP may occur by increased activity ofthe MMP inhibitors (TIMP). We have verified the increased expression ofTIMP in PD tissue using the more sensitive RT/PCR procedure, whichshowed a 2-fold stimulation of TIMP1. The increased expression of TIMP1should lead to an increased inhibition of MMP.

Example 13. The Expression of a Family of Wound Healing-RelatedPeptides, the Thymosins, is Increased in Human PD

In the DNA microarray study mentioned above, we found increasedexpression of peptides belonging to the thymosin-β family (Table 1).These proteins stimulate MMP activity, cross-link to fibrin andcollagen, and promote wound healing (Huff et al., 2002; Malinda et al.,1999; Sosne et al., 2002). Their increased synthesis in the PD plaquemay be another manifestation of a defense mechanism that is unable tocontrol or arrest the progression of fibrosis. The administration ofthymosin-β4 has been proposed for wound healing (Huff et al., 2002;Malinda et al., 1999; Sosne et al., 2002), and, as stated above, PD islikely the result of an injury that does not heal properly. It should bepossible to further up-regulate this endogenous defense mechanism bypharmacologically increasing thymosin levels in the TGF-β1-inducedlesions in the rat model of PD.

Example 14. Investigating the Role of NO and ROS in PD Using the iNOSKnockout Mouse Model

The blockade of iNOS activity in the rat by long-term oral L-NILadministration (Ferrini et al., 2002) is only partially effective ininhibiting iNOS. Therefore, the iNOS knockout mouse (Hochberg et al.,2000) is of use for studies where iNOS expression is completely absent.NO in this animal model can be synthesized only by the other NOSisoforms, namely eNOS and nNOS. These isoforms are in generalconstitutive and as such are difficult to induce. It therefore seemsunlikely that they would play a significant anti-fibrotic role. The iNOSknockout mouse has previously been used to show that experimentalurethral fibrosis is intensified as a consequence of the iNOS knockout(Tanaka et al., 2002). We have tested whether it is possible to developa PD-like plaque by injection of TGF-β1 into the TA, injecting 0.2 ug ofTGF-β1 into the TA of a wild type mouse. FIG. 11 shows plaque formationwithin the TA at 6 weeks as shown with Masson staining.

Example 15. Therapeutic Intervention in PD

The results above demonstrate that PDE5 and 4 are both expressed in thehuman and rat normal tunica albuginea, and the respective PD and PD-likefibrotic plaques, as well as in the cell cultures obtained from thesetissues. The results also demonstrate the inhibition of a TGF-1-inducedfibrotic plaque in the rat model of PD, through the reduction ofcollagen deposition and possibly an increase in apoptosis of theresident fibroblasts and myofibroblasts, by long-term oraladministration of the respective PDE5 and cAMP-dependent PDE inhibitors,sildenafil and pentoxifylline, and the NOS substrate, L-arginine.

The in vitro effects of both PDE inhibitors and a cGMP analog, 8Br-cGMP, on fibroblast cultures obtained from the human PD plaque,indicate that these agents may be effective against fibrosis by reducingthe relative number of fibroblasts/myofibroblasts through the inductionof apoptosis of these cells. We also found that these compounds a)interfere with fibroblast differentiation into myofibroblasts, the cellsthat are key players in tissue fibrosis, and b) down-regulate thesynthesis of collagen I but not collagen III. The effects of sildenafilmay be exerted through the inhibition of PDE-5, and in the case ofpentoxifylline through a cAMP-dependent PDE, potentially PDE4. Theresults open a new approach for the treatment of PD and, by extension,tissue fibrosis, based on the use of PDE inhibitors and other enhancersof PDK activity, and possibly of compounds and biologicals that enhanceNO synthesis.

The reduction of the fibrotic plaque observed in vivo in animalsreceiving L-arginine, coincides with its effects in preventingexperimental ethanol-induced inflammatory and fibrotic changes in liver,kidney, lung, and cardiovascular system (Nanji et al., 2001; Peters etal., 2000; Simko and Simko, 2000; Susic et al., 1999; Song et al., 1998;Bing et al., 2002; Alves et al., 2002). The action of L-arginine may bemediated by the stimulation of NOS activity. This was previously shownby the increase of L-arginine levels in the penis and the improvement oferectile dysfunction in the aging rat by NOS stimulation achieved aftera regimen of L-arginine administration of 2.2 g/kg/day (Moody et al.,1997). This dose is within the range normally employed asvasculoprotective for long-term studies in the rat (Bing et al., 2002;Alves et al., 2002).

The in vivo and in vitro results showing an inhibition of collagensynthesis and stimulation of apoptosis in the PD-like plaque and in PDcells by both sildenafil and pentoxifylline, are in good agreement withthe extensive use pentoxifylline as an antifibrotic agent in liver andvascular fibrosis (Becker et al., 2001; Raetsch et al., 2002; Chen etal., 1999; Tarcin et al., 2003). The fact that the cGMP analog 8-Br-cGMPinhibited collagen I synthesis and induced apoptosis in PD cellssuggests that in the case of sildenafil the in vivo effects on thefunction of the fibroblasts/myofibroblasts in the TA may be mediated bythe elevation of cGMP levels. In addition, cGMP analogs, PKG activators,and PDE inhibitors have been shown to inhibit collagen synthesis(Redondo et al., 1998; Wollert et al., 2002), and induce apoptosis(Sirotkin et al., 2000), and some of the PDE inhibitors like sulindacsulfone (Exisulind) are effective as anticancer agents because of theirintense pro-apoptotic action (Piazza et al., 2001; Thompson et al.,2000). However, since pentoxifylline did not affect cGMP levels in thehuman PD fibroblasts, and the drug is considered to be a non-specificinhibitor of cAMP-PDE (Lin et al., 2002c; Liang et al., 1998), and atleast in some cell types does not affect cGMP levels (Chen et al.,1999), the increase in cAMP may also have played a role in theantifibrotic effects observed with pentoxifylline. Whether this occursvia the inhibition of PDE4 present in TA and PD remains to beestablished. Pentoxifylline may also act through its blockade ofPDGF-induced activation of the mitogen activated protein kinase system(Souness et al., 2000) and of other cytokine-mediated fibrogenicmechanisms (Raetsch et al., 2002).

The daily dose of pentoxifylline used was ⅕ of the oral dose normallyemployed in rats for the long-term treatment of fibrosis (Chen et al.,1999; Tarcin et al., 2003), and in the case of sildenafil, it is ½ to1/7 of the chronic dosage used in recent studies in rats (Sebkhi et al.,2003). When the 10 mg/kg/day dose is translated into the equivalent dosein humans by correcting for differences in the body weight/skin area(Freireich et al., 1966), it is roughly 1.5 mg/kg which is about thedose ingested by men with an on demand single 100 mg tablet. Theselected dose was dispensed in 24 hours and not as a bolusadministration, so that concentrations at a given time should be muchlower, considering the short half-life (about 4-6 hours) of sildenafil.Therefore, the daily doses of the PDE inhibitors tested in the currentwork are not supra-pharmacological or associated with toxicity. Inaddition, it is possible that local administration of either L-arginineor the PDE inhibitors, e.g. by injection into the plaque or in vehiclesable to traverse the skin and TA may considerably reduce the effectivedosage.

It is unknown why administration of L-arginine, which should increase NOsynthesis and hence cGMP levels and has been shown to be effective inarresting the growth of the TGF-B1 induced plaque in the rat model ofPD, failed to stimulate apoptosis, as could be expected from its effectsincreasing it in vivo in the smooth muscle of the pulmonary arteries(Wang et al., 1999; Holm et al., 2000). However, the absence of astimulation of the apoptotic index in the PD plaque by L-arginine mayagree with the decrease in apoptosis observed in liver transplants whichis in line with the anti-apoptotic effects of NO in certain conditionsand tissues (Wang et al., 2002b). In any case, not only cGMP but itsdown-stream compound in the NO-cGMP cascade, PKG, is also effective inpreventing fibrosis and remodeling in balloon-injury and arterialrestenosis (Wollert et al., 2002; Chiche et al., 1998), as shown by genetransfer of the PKG cDNA in rats.

The results demonstrating the presence of PDE5A and PDE4 in the TA andPD plaque in the human and rat, and in their respective fibroblastcultures, provide a rationale for the anti-fibrotic effects of PDEinhibitors on the PD animal model and on the PD cell cultures. ThePDE5A1 and PDE5A2 proteins have been previously localized in humanpenile corpora cavernosa (Lin et al., 2000a). The PDE5A3 variant wasalso found in corpora cavernosa and confined to tissues with a smoothmuscle or cardiac muscle component, and is twice as sensitive as PDE5A1to sildenafil, but, as with PDE5A1 and 2, is subject to transcriptionalup-regulation by both cAMP and cGMP (Lin et al., 2002a; Turko et al.,1999). As to PDE4, cAMP can activate PKG nearly as effectively as cGMP,so that eventually, the inhibition of PDE4 may cause PKG effects (e.g.,counteracting fibrosis) similar to those exerted by as the inhibition ofPDE5A.

The results reported above indicate that pharmacological interventionsaimed at elevating NO, cGMP, or PKG levels, and possibly cAMP, in thepenis are of use for the treatment of PD, and potentially, for otherfibrotic conditions. This work has not addressed the question on whetherintervention would induce regression of an already well-formed plaque,but comparison of multiple gene expression profiles in human PD and therelated Dupuytren's disease suggest that both conditions are in adynamic cell and protein turnover involving replication,differentiation, apoptosis, and collagen and extracellular matrixsynthesis and breakdown (Magee et al., 2002b; Gonzalez-Cadavid et al.,2002; Gholami et al., 2002). Therefore, modulation of any of theseprocesses may involute the plaque, as has been observed in generalizedfibrotic conditions (Lee et al., 2001; Lai et al., 2000).

Example 16. Intensification of Aging-Related Fibrosis in the ArterialMedia by iNOS Inhibition

In order to determine whether aging per se is associated with anintensification of collagen deposition and a relative loss of SMC in themedia from the aorta to the peripheral resistant arteries(Breithaupt-Grogler and Belz, 1999; Robert, 1999; Integan and Schiffrin,2000), staining was performed on sections from the abdominal aorta,femoral, and brachial arteries as well as from the penile shaft focusingon two peripheral putative resistance arteries: the bulbourethral anddorsal arteries of the penis. FIG. 15A shows that in the media of thedorsal penile artery, few collagen fibers were present in the young ratsbut were considerably increased in the aged animals, resembling thesituation seen in the aorta. Consistent with the model that iNOS may actas antifibrotic agent within the vascular tree, the administration ofL-NIL, a specific inhibitor of iNOS activity, for 3 weeks to the agedrats led to a further increase in the collagen fibers within the mediaof the aorta and the dorsal penile artery.

Image analysis was performed in all arteries with the exception of thebrachial (FIG. 15B), on 5 animals per experimental group, and 6 sectionsper animal (3-4 fields per section). In all vessels studied, there was amarked reduction in the SMC/collagen ratio with aging. Following iNOSblockade by L-NIL, there was a further exacerbation in the amount ofcollagen within the media (with the exception of the bulbourethralartery), suggesting that the decrease in NO production by the inhibitionof iNOS leads to an intensification of the aging-related fibrosis. Thesealterations were not accompanied in the resistant arteries by asignificant increase in the intima/media thickness (IMT), whereas in theaorta and femoral the IMT was higher (Table 2). The measurements of theluminal diameter (Table 2) confirmed the clinical observation that thedorsal and bulbo-urethral arteries, with a luminal diameter well below350 um, fall within the definition of resistance arteries (Intengan andSchriffin, 2000; Moore and Schiffrin, 2001).

Example 17. iNOS Induction and Peroxynitrite Deposition in the ArterialMedia with Aging

All the antibodies used in this work have been validated (Ferrini etal., 2001a, 2002; Vernet et al., 2002; Goettsch et al., 2001), and inthe case of the iNOS antibody it was additionally tested in the currentwork by immunocytochemistry against rat fibroblast cultures (peniletunica albuginea, see Ferrini et al., 2002; Vernet et al. 2002) inducedto express iNOS with a cytokine cocktail, producing 130 uM nitrites(Hung et al., 1995). These cells were intensively stained, in comparisonto uninduced cells (<10 uM nitrites) that were negative (not shown).Western blots revealed the expected single 130 kDa band in extracts ofthe induced cells, also detected by the corresponding monoclonalantibody, and this band was absent in aorta extracts from a young iNOSknockout mouse that had received LPS (4 mg/kg) to induce iNOS, whereasthe band was visible in the respective extract from the similarlytreated wild type animal (not shown).

This antibody showed that iNOS is increased with aging in parallel withcollagen deposition in the arterial media throughout the vascular tree,confirmed by detection of nitrotyrosinylated proteins. The latter arisefrom peroxynitrite produced by the reaction between NO and ROS, andtherefore are an indirect measure of NOS activity. FIG. 16A showsnegligible iNOS expression and nitrotyrosine formation in the dorsalartery of the penis of the young animals, and a remarkableintensification of both processes with aging. The iNOS staining in thesevessels was mainly confined to the media and intima. A similar findingwas seen in the aorta, brachial, and femoral arteries (not shown). WhenL-NIL, the inhibitor of iNOS activity, was given, there was a reductionin iNOS expression, which combined with the direct decrease of iNOSactivity, led to a reduction in peroxynitrite formation. Quantitation byimage analysis confirmed these changes in the resistant dorsal andbulbourethral arteries of the penis (FIG. 16B), as well as in the aortaand femoral arteries (not shown). The brachial artery was not subjectedto image analysis.

Example 18. Effects of iNOS Inhibition on ROS Production, Apoptosis, andPAI in the Arterial Media

Utilizing the Cu/Zn SOD as an indirect marker of ROS, the production ofROS in the arterial media was found to be considerably increased withaging in the femoral, brachial and resistant arteries but not in theaorta, and this process was further increased with iNOS inhibition byL-NIL (FIG. 17A). This was additionally confirmed by image analysis(FIG. 17B), that indicated that L-NIL blockade of iNOS activity raisedCu/Zn SOD by 40 to 50%. The Mn SOD gave similar results from the aortato the resistant arteries (not shown). Another antioxidant enzyme, hemeoxygenase I, demonstrated the same aging related changes in the peniledorsal artery, as observed for both SOD enzymes, but remarkably, L-NILdid not induce a further significant change in the expression of thisenzyme (FIG. 18). The localization of virtually all the expression ofheme oxygenase-1 was in the arterial adventitia, rather than in themedia as seen for the SOD enzymes.

The NOROS balance was significantly altered throughout the entirearterial media by iNOS inhibition with L-NIL via a reduction in NOsynthesis (denoted by peroxynitrite) and a stimulation of ROS formation(denoted by the antioxidant enzymes). Apoptosis of the SMC within themedia of the penile resistance arteries increased with aging, anddecreased subsequently in the old animals receiving L-NIL treatment(FIG. 19A). The apoptotic index was calculated for both the dorsal andbulbourethral penile arteries by image analysis, and was higher in agedcompared to young rats but L-NIL treatment resulted in a reduction inthis index (FIG. 19B).

Aging alone or in combination with iNOS inhibition affected theexpression of PAI-1, a well characterized inhibitor ofmetalloproteinases (Li et al., 2000; Kaikita et al., 2002). Inhibitionof PAI is associated with an increase in collagen fibers due to itsinterference with metalloproteinases that are involved with thebreakdown of collagen. Compared to young animals, PAI expression wasconsiderably increased in the arterial media with aging, and was evenfurther stimulated by iNOS inhibition, as seen in the resistant artery(FIG. 20A). Quantitative image analysis for both the mean intensity ofexpression (FIG. 20B) and the number of PAI positive cells (FIG. 20B)indicated that the increase in PAI by aging alone was between 2- and5-fold, respectively. However, the effect of L-NIL on PAI expression inthe aged media was negligible (FIG. 20B).

These results indicate that the arterial media from the aorta to thesmall resistant arteries undergoes many of the changes that occur withinthe corporal tissue with aging, namely: a) a reduction in theSMC/collagen ratio; b) an increase in markers of oxidative stress, andof inhibitors of collagen degradation, such as PAI, which are knownpro-fibrotic factors; and c) the spontaneous induction of iNOS, which isbelieved to act as an anti-fibrotic agent (Vernet et al., 2002; Ferriniet al, 2002; Hochberg et al, 2000). However, the increase in SMCapoptosis in the media of the resistant arteries of the penis,presumably leading to a reduction in the absolute SMC content, contrastswith what has been reported for large vessels such as the aorta and thefemoral artery (Connat et al., 2001; Asai et al., 2000), but does agreewith the process described in the corporal SMC (Garban et al., 1995;Ferrini et al., 2001a).

Our results also confirm the role for NO derived from iNOS produced bythe SMCs of the media in combating aging-related fibrosis within themedia, as evidenced both by the increase in ROS and an intensificationof fibrosis within the media of the arterial wall when iNOS activity isinhibited. The excessive deposition of collagen fibers observed in thearterial media of the aged rats is thought to lead to arterial stiffnessor arteriosclerosis in the vascular system. Because of the apoptosisoccurring in the SMC, the relative reduction in the SMC/collagen ratiois intensified in the resistant arteries of the penis, in comparison tothe larger arteries, e.g. the aorta. This process may be a primaryfactor involved in the development of essential hypertension, which isvery prevalent with aging.

In the case of the penile arteries, the data also indicate that areduction in the ability of penile vessels to relax normally duringcavernosal nerve stimulation leading to an erection, may contribute inpart to the high prevalence of ED associated with aging. In addition tothis aging-related fibrosis of the arterial media (Breithaupt-Groglerand Belz, 1999; Robert, 1999; Integan and Schiffrin, 2000; Fornieri etal., 1992), it is well documented (Grein and Schubert, 2002) thatsimilar fibrotic changes occur within the penile corporal sinusoids. Thecorporal tissue comprises primarily of a syncytium of vascular SMC withan endothelium lining which is biologically and physiologicallyindistinguishable from the one present in the media and intima of thevascular tree (Krall et al., 1988) and may be considered a highlyevolved extension of these arterial tissues. Therefore, insults thatafflict the arterial media may also afflict the corporal SMCs, resultingin defective vaso-relaxation in both the corporal tissue (ED) and thearterial tree (hypertension). Indeed, the prevalence of ED andhypertension in man seems to parallel each other as a function of age(Sullivan et al., 2001; Melman and Gingell, 1999), and many disordersthat damage one of these vascular tissues also seem to impact the othere.g. diabetes, chronic renal failure, etc. In all these disorders,vascular oxidative stress and fibrosis, leading to arteriosclerosis, arecommon denominators at the histological and molecular and levels.

The results on the abdominal aorta and the rest of the smaller arteriesand arterioles are in agreement with previous studies from other groupsshowing in the aging rat both an intensification of oxidative stress(van der Loo et al., 2000; Demaree et al., 1999) and collagen deposition(Goettsch et al., 2001; Chou et al., 1998; Csiszar et al., 2002), thatis the likely cause of the reduction of the SMC/collagen ratio withinthe media. This alteration, that in the large vessels does not appear tobe caused by SMC apoptosis (Connat et al., 2001; Asai et al., 2000),would explain the clinical observation in humans of diminished arterialelasticity associated with aging, which in some instances is compoundedby a reduction of the arterial lumen due to media/intimal thickening(Moore and Schiffrin, 2001). The fact that different vessels in thearterial tree, regardless of size or location, seem to experiencefibrosis of the media may explain dysfunctional vasorelaxation orimpaired perfusion of many organs that occurs with aging(Breithaupt-Grogler and Belz, 1999; Robert, 1999; Integan and Schiffrin,2000; Fornieri et al., 1992; Garban et al., 1995; Ferrini et al., 2001a;Rogers et al., 2003; Berry et al., 2001). The ability of the resistantarteries to relax normally is fundamental for the control of thesystemic blood pressure, and as exemplified by the dorsal penile andbulbourethral arteries in this study, they showed an intensification ofSMC loss due to apoptosis without a change in IMT, which agrees with hasbeen previously reported for the mesenteric small resistant arteries inhypertension (Rizzoni et al., 2000).

Although NO has been shown in animal models to be protective againstatherosclerosis and restenosis in the vascular system (Gewaltig andKojda, 2002; Cheng et al., 2001), and fibrosis throughout the vasculartree and other organs (Ferrini et al., 2002; Vernet et al., 2002;Gewaltig and Kojda, 2002), the concept that NO may prevent aging-relatedarteriosclerosis is novel. In fact, the pro-apoptotic action of NO(Gewaltig and Kojda, 2002; Kibbe et al., 1999) would suggest that itdecreases the SMC/collagen balance through increased cell death. We havefound in the aged animals treated with L-NIL an association between NOSinhibition and subsequent reduction of nitrotyrosine formation, with adecrease of apoptosis, which would suggest that NO does cause some SMCloss in the penile resistant arteries similar to what has beenpreviously assumed to occur in the corpora cavernosa (Ferrini et al.,2001a). However, an increased apoptotic index may be balanced by astimulation of cell replication or tissue remodeling (Ingengan andSchiffrin, 2001), and what really matters physiologically is the netbalance between both processes. In the data above, the relative numberof SMC in the arterial media (represented by the smooth muscle/collagenratio) was severely reduced when NO synthesis was diminished by L-NIL.This, together with the well known effects of NO in scavenging theprofibrotic compound, ROS, thereby decreasing collagen synthesis anddown-regulating its breakdown (see Ferrini et al., 2002; Vernet et al.,2002), would support the view of an overall beneficial role of NO inpreventing arterial stiffness and loss of compliance of the corporacavernosa.

A final question is whether collagen accumulation with aging is at leastpartially mediated via the regulation of PAI-1, TIMP1 and othermetalloproteinase inhibitors (Li et al., 2000; Kaikita et al., 2002),that increase in different types of fibrosis. The current results withPAI, combined with previous data where we observed considerablemetalloproteinase and PAI mRNA expression in the fibrotic plaque ofPeyronie's disease in the human and rat (Magee et al., 2002a), wouldsuggest that although the increase in the pro-fibrotic PAI may induce acompensatory elevation of metalloproteinase levels, the enzyme wouldremain inhibited and the net result would be an impaired collagenbreakdown.

In conclusion, the results indicate that within the arterial system andthe cavernosal tissue it may be possible to pharmacologically modulatea) the NO/ROS balance with NO donors or other NO generators togetherwith antioxidants, and b) the PAI/MMP balance with agents modifyingtheir relative expression. Such novel therapies may constitute viableapproaches for the prevention and/or therapy of vascular disorders thatinvolve the arterial media and the corpora.

Example 19. Gene Therapy with iNOS cDNA

All studies throughout this section, unless specifically indicated, areperformed in the rat model where the PD-like plaque is initiated by theinjection of TGF-β1 (0.5 ug) into the TA. TGF-β1 transcriptionallyamplifies its own synthesis, allowing for a single injection.Saline-injected TA are used as controls. The AdV-CMV-iNOS construct hasbeen prepared by subcloning the iNOS cDNA driven by the strong CMVpromoter (Garban et al., 1997), from a plasmid construct into an AdVplasmid vector, and purifying the AdV construct, as previously describedfor PnNOS (Magee et al., 2002a). This AdV vector isreplication-defective and helper-dependent, and therefore isnon-infectious and totally innocuous. In addition, it lacks virtuallyall the original viral sequences that may be immunogenic. This AdVconstruct can be transfected into the TA, and it has been cloned andutilized for other therapeutic purposes in the penis (Magee et al.,2002a).

Rats are injected in the TA at the same site as the TGF-β1 was injected5 days earlier (as evidenced by a non-absorbable suture) with 10⁸ and10⁹ vp of either AdV-CMV-iNOS in 50 ul saline, or with vehicle (saline)only. This 5-day waiting period between the TGF-β1 injection and thecDNA construct avoids any interference of the viral preparation with theinjected TGF-β1, and/or its dispersion by the electroporation applied toenhance transfection of the iNOS construct. The resulting 4 groups ofrats are allowed to develop the plaque for 40 more days, sacrificed, andthe area around the plaque is excised, fixed, paraffin-embedded, andsectioned (Ferrini et al., 2002, Vernet et al., 2002). In another 2groups of rats injected with TGF-β1 to induce a plaque, the cDNAconstruct and saline are injected 45 days after the TGF-β1 injection(when the plaque has already formed) and 30 days later the animals aresacrificed.

In the “early” treatment groups (iNOS given 5 days after TGF-β1injection), the TA of the iNOS-treated animals shows, in comparison withcontrols: 1) a decrease in the size of the plaque as evidenced byMasson, collagen I/III staining, and hydroxyproline content: 2) a higherexpression of iNOS and nitrotyrosine; 3) decrease of ROS; and 4)increase in the apoptotic index of the fibroblasts/myofibroblasts. Inthe “late” treatment group, where the iNOS is injected 45 days afterTGF-β1 injection, at least some regression of the plaque is obtained.

Example 20. Oral NO Donors or NOS Substrate

Plaques are induced in the TA of rats by TGF-β1 injection (Ferrini etal., 2002). Drinking water containing molsidomine(N-ethoxycarbonyl-3-morpho-linosydnomine), at 0.12 g/l (Benigni et al.,1999) (freshly prepared each day) is given to 2 groups of rats, an earlyand a late treatment group. The dose of molsidomine used is based on thereport in which it was utilized for 22 days to protect againsttubulo-interstitial injury in a rat model of chronic glomerular disease(Uckert et al., 2001), and is calculated to be equivalent toapproximately 15 mg/kg/day in the rat. In the case of L-arginine, it isgiven only as a late treatment, but at 2 doses: 22.5 and 10.0 g/l (indrinking water) (2 groups). Previously, the 22.5 g/l dose of L-argininewas used for 45 days to elevate NOS activity in the rat penis (Moody etal., 1997), and to inhibit the plaque with the early treatment isequivalent to roughly 2.8 g/kg/day.

Example 21. Oral PDE Inhibitors to Regress the PD Plaque

Sildenafil (specific PDE5 inhibitor), and pentoxifylline (non-specificPDE inhibitor), are given at doses of 100 mg/I in the drinking water, aswell as a control (drinking water only), beginning on day 45 (3 groups).The study may be repeated at double or possibly quadruple the dosageabove (2 groups). Reduction in plaque size was observed with bothsildenafil and pentoxifylline during the entire time of plaquedevelopment (‘early treatment’), and this type of treatment may be aseffective in regressing the plaque (‘late treatment’).

Eleven PDE mRNAs for the respective isoforms have so far been identifiedby RT/PCR in the human (Kim et al., 2000). Due to the fact thatpentoxifylline is more effective than sildenafil at inhibiting apoptosisin the PD plaque, it is likely that more than one PDE gene product maybe involved in plaque development, and the pure PDE4 inhibitor, rolipram(Uckert et al., 2001) may identify a related, relevant PDE. The increasein cAMP may act directly, or through stimulating the synthesis of cGMP(Kim et al., 2000). Rolipram, is given to early and late treatmentgroups (2 groups), at the same dose, in order to determine whetherincreasing cAMP levels is as, or more, effective than cGMP.

Example 22. Gene Therapy with PKG

The AdV-PKG (wild type) and AdV-PKGcat (mutated) are injected into theTA(2 groups). The constitutively active PKG1 mutant consists of thecarboxy-terminal catalytic domain without the amino-terminal regulatorydomain where cGMP binds (Wollert et al., 2002). Both the wild type andmutated constructs are obtained from Dr. Stefan Janssens (Center forTransgene Technology and Gene Therapy, University of Leuven, Belgium).

Example 23. Screening of Other cGMP-Dependent PDE Isoforms

Tissues from human normal TA and PD plaque preserved in RNA later (Mageeet al., 2002b; Ferrini et al., 2002) or the previously isolated RNAsfrom these tissues, conserved at −80° C., are used. In the case of therat model, a 2-3 mm transverse section is obtained at the site of salineor TGF-β1 injection 45 days after plaque initiation. RNA is isolatedfrom 2 groups of human and 2 groups of rat tissues.

Example 24. Oral Antioxidant

The antioxidant vitamin E (α-tocopherol) is given in a speciallyprepared oral diet, so that the animal receives in an early treatmentphase approximately 200 IU/kg/bw/day, whereas controls receive thenormal diet containing less than 20 IU/kg (Gonca et al., 2000). Thethird group (3 groups) receive twice a day an intramuscular injection(10 mg/kg) of another antioxidant compound that ameliorates oxidativestress and lipid peroxidation, the glutathione precursorS-adenosyl-L-methionine (SAM) (Muriel et al., 1998b). Depending on whichantioxidant is more efficacious in the early treatment, a late treatmentbeginning on day 45 after TGF-β1 injection and lasting for 30 days isperformed with the selected compound compared to a control group withnormal diet (2 groups).

Example 25. Effect of Obliterating iNOS on Collagen Breakdown. Plaque inthe iNOS Knockout

The PD-like plaque is induced with TGF-β1 given into the TA of the iNOSknock-out mouse, utilizing the wild type mouse as a control, (2 groups,n=6). 45 days after the injection of TGF-β1, the mice are sacrificed andthe tunical tissues is either fixed and sectioned forimmunohistochemistry (n=3) or used for RNA isolation (n=3). Theexperiment is repeated, but this time, 8 days before sacrifice, thecollagen I promoter plasmid is injected and electroporated (Magee etal., 2002a) (2 groups; n=3). Animals are sacrificed and fresh tunicalplaque tissue obtained for β-galactosidase expression and zymography.

Example 26. Modulation of MMP Through Thymosin Peptides. Treatment withThymosin β

Thymosin β4 and 10 are given daily intraperitoneally as a late treatmentat 60 ug/day, every other day (2 groups) (Sosne et al., 2002). Suchtreatment with thymosin-β4 (the most abundant thymosin) has been usedfor promoting healing of dermal wounds (Sosne et al., 2002)). Plasmidpreparations of a cDNA encoding both peptides (200 ug/rat) are alsogiven by injection/electroporation to the TA (2 groups), as a latetreatment (i.e. 45 days after the TGF-β1 injection, to induce the PDlike plaque).

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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TABLE 1 Dupuytren Peyronie Protein/gene n* Mean ± SE n** Mean ± SEmatrix 9 29 ± 10 2 4.7 ± 2.6 metalloproteinase 2 matrix 2 50.8 ± 0.8 metalloproteinase 9 thymosin beta-10 9 5.9 ± 2.6 5 5.5 ± 1.3 (TMSB10)thymosin beta 4 8 5.9 ± 1.5 5 2.5 ± 0.9 prothymosin alpha 2 2.6 ± 0.0 26.2 ± 3.8 osteoblast specific 5 5.6 ± 1.4 3 4.3 ± 0.5 factor 1 (OSF-1)osteoblast specific 4 26.7 ± 12.7 factor 2 (OSF2) rho GDP dissociation 63.5 ± 1.4 2 18.3 ± 2.4  inihibitor 1 (RHO-GDI 1) n* = 9 patients n** =10 patients

TABLE 2 YOUNG OLD OLD + L-NIL (μm) (μm) (μm) INTIMA MEDIA THICKNESSAORTA  73.4 ± 8.6**  93.0 ± 6.6** 85.2 ± 6.6 FEMORAL 49.7 ± 7.1 63.5 ±2.4 51.8 ± 4.6 PENILE DORSA 17.0 ± 2.3 18.6 ± 1.4 23.6 ± 3.3 BULBOURETHRAL 10.5 ± 1.3 10.1 ± 1.6  9.9 ± 0.7 LUMEN DIAMETER PENILE DORSAL100.8 ± 13.7 118.7 ± 6.6  105.5 ± 16.  BULBO URETHRA 40.1 ± 5.6 42.6 ±6.8 47.3 ± 6.6

1. A method comprising administering a cyclic guanosine 3′, 5′-monophosphate (cGMP) type 5 phosphodiesterase (PDE-5) inhibitor according to a continuous, long-term regimen to an individual with at least one of a penile tunical fibrosis and corporal tissue fibrosis, wherein the PDE-5 inhibitor is selected from the group consisting of sildenafil, tadalafil, and vardenafil, wherein the PDE-5 inhibitor is administered at a dosage of up to 1.5 mg/kg/day, and wherein the PDE′″5 inhibitor is administered for greater than or equal to 45 days.
 2. The method of claim 1, wherein the PDE-5 inhibitor is sildenafil.
 3. The method of claim 1, wherein the PDE-5 inhibitor is tadalafil.
 4. The method of claim 1, wherein the PDE-5 inhibitor is vardenafil.
 5. The method of claim 1, wherein the individual has penile tunical fibrosis.
 6. The method of claim 1, wherein the individual has corporal tissue fibrosis.
 7. The method of claim 5, wherein the penile tunical fibrosis is Peyronie's disease
 8. The method of claim 6, wherein the penile corporal fibrosis is corporal veno-occlusive dysfunction.
 9. The method of claim 1, wherein the PDE-5 inhibitor is administered orally, by injection, or by local administration to a penis.
 10. The method of claim 9, wherein the PDE-5 inhibitor is administered orally.
 11. The method of claim 1, wherein administering according to a continuous, long-term regimen comprises administering for months.
 12. The method of claim 1, wherein administering according to a continuous, long-term regimen comprises administering for years.
 13. The method of claim 1, wherein administering according to a continuous, long-term regimen comprises administering at least once per day.
 14. The method of claim 1, wherein administering according to a continuous, long-term regimen comprises administering in a single dosage once per day.
 15. The method of claim 1, wherein the PDE-5 inhibitor is constantly present in the individual during the continuous, long-term regimen.
 16. The method of claim 1, wherein the PDE-5 inhibitor administration arrests, reduces, or regresses the penile tunical fibrosis in the individual.
 17. The method of claim 1, wherein the PDE-5 inhibitor administration arrests, reduces, or regresses the corporal tissue fibrosis in the individual.
 18. The method of claim 1, wherein the PDE-5 inhibitor is tadalafil orally administered to the individual once per day for months.
 19. A method comprising orally administering tadalafil to an individual with at least one of a penile tunical fibrosis and corporal tissue fibrosis, wherein tadalafil is administered at least once per day at a dosage of up to 1.5 mg/kg, and wherein tadalafil is administered for greater than or equal to 45 days.
 20. The method of claim 19, wherein the individual has penile tunical fibrosis.
 21. The method of claim 19, wherein the individual has corporal tissue fibrosis.
 22. The method of claim 20, wherein the penile tunical fibrosis is Peyronie's disease.
 23. The method of claim 21, wherein the penile corporal fibrosis is corporal veno-occlusive dysfunction.
 24. The method of claim 19, wherein tadalafil is administered for months.
 25. The method of claim 19, wherein tadalafil is administered for years.
 26. The method of claim 19, wherein the tadalafil is orally administered to the individual once per day for months.
 27. The method of claim 19, wherein the tadalafil is constantly present in the individual during the administration.
 28. The method of claim 19, wherein tadalafil administration arrests, reduces, or regresses the penile tunical fibrosis in the individual.
 29. The method of claim 19, wherein tadalafil administration arrests, reduces, or regresses the corporal tissue fibrosis in the individual.
 30. A kit comprising a PDE-5 inhibitor and instructions for administering the inhibitor at a dosage of up to 1.5 mg/kg/day to an individual with at least one of a penile tunical fibrosis and corporal tissue fibrosis according to a continuous, long-term regimen of greater than or equal to 45 days. 